TBSD#2262589, VOL 0, ISS 0

Miraculin-based sweeteners in the protein-engineering era: an

alternative for developing more efficient and safer products
Rafael Trindade Maia, Iv^ania Samara dos Santos Silva, Adeilma Fernandes de Souza, Nilton Ferreira
Fraz~ao, Rafael Medeiros de Lima, and Magn olia de Ara
ujo Campos

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 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
1 https://doi.org/10.1080/07391102.2023.2262589 60
2 61
3 REVIEW ARTICLE 62
4 63
5 Miraculin-based sweeteners in the protein-engineering era: an alternative for 64
6 65
7 developing more efficient and safer products 66
8 67
9 Q6 Rafael Trindade Maia b
a,b
, Iv^ania Samara dos Santos Silvab, Adeilma Fernandes de Souzab, 68
10 Nilton Ferreira Fraz~ao , Rafael Medeiros de Limab and Magno lia de Arau
jo Camposb 69
11 a
Center for Sustainable Development of Semiarid, Federal University of Campina Grande, Sume, Brazil; bPost-Graduation Program in Natural 70
12 Science and Biotechnology, Center of Education and Health, Federal University of Campina Grande, Cuite, Brazil 71
13 Communicated by Ramaswamy H. Sarma 72
14 73
15 74
16 ABSTRACT ARTICLE HISTORY 75
17 The current sweeteners available are very efficient in providing sweet taste. However, they are associ- Received 21 March 2023
Accepted 16 September 2023
76
18 ated with several chronic diseases. Some glycoproteins, such as miraculins, are extremely interesting
from a biotechnological point of view because they perform the bitter into sweet taste modifying
77
19 function excellently, in addition to being safer as food. In contrast, purifying and synthesizing these KEYWORDS 78
20 proteins represents a major challenge for the food industry, as these proteins are large and complex
Glycoproteins; food
additive; peptide design;
79
21 molecules, which would make the final product expensive and economically unviable. In this context, computational biology; 80
22 emerging techniques from computational biology and molecular modelling have been promoting a molecular modelling 81
23 remarkable revolution in protein bioengineering. Bioinspired peptides can provide many possibilities
in sweeteners development through rational design. Once these peptides are smaller molecules than
82
24 an entire protein, its synthesis on a large scale tends to be much easier and more economical, besides 83
25 presenting a potential for better bioavailability in the organism. The techniques discussed here allow, 84
26 through sophisticated pipelines and algorithms, to perform the rational design of mimetic peptides 85
27 and with smaller size, which can carry out the activation of sweet taste of miraculins and to be more 86
28 viable for industrial production. In this review, the premises and tools for the elaboration of synthetic
peptides bioinspired in proteins with sweetening activity that mimic this action will be emphasized.
87
29 88
30 89
31 90
32 Introduction exploration and utilization of various bioactive molecules 91
33 Bioactive molecules as sweeteners
sourced from plants, fruits, and other natural sources as 92
34 potential sweeteners. 93
35 The main bioactives are chemical compounds present in vari- In a study by the authors investigated the sweetening 94
36 ous foods and natural products that can perform important potential of steviol glycosides extracted from the leaves of 95
37 biological functions in the human body. Among these mole- Stevia rebaudiana (Smith et al., 2022) The research demon- 96
38 cules, sweeteners are a relevant group, as they have been strated that steviol glycosides not only possess intense 97
39 widely used as sugar substitutes in foods and beverages, sweetening properties but also exhibit a minimal impact on 98
40 seeking to meet the needs of individuals who aim to reduce blood glucose levels, positioning them as promising candi- 99
41 caloric intake or avoid sugar consumption for health reasons. dates for addressing concerns related to diabetes and obes- 100
42 One of the widely studied and used sweeteners is aspartame, ity. Similarly, an study explored the sweetening capabilities 101
43 a dipeptide composed of phenylalanine, aspartic acid and of mogrosides extracted from Siraitia grosvenorii, also known 102
44 methanol. Aspartame, for example, has significantly greater as monk fruit (Chen et al., 2021). The researchers revealed 103
45 sweetening power than regular sugar and is found in a var- that mogrosides not only offer a natural sweetness but also 104
46 iety of commercial products, including diet drinks, confec- present antioxidant properties, further contributing to their 105
47 tionery and low-calorie foods. appeal as a potential sugar substitute. 106
48 Bioactive molecules, especially those with sweetening Continuing this trend, a work delved into the sweetening 107
49 properties, have garnered significant attention in the field of effects of thaumatin, a protein derived from the katemfe fruit
108
50 food science and nutrition due to their potential to provide (Thaumatococcus daniellii) (Garcia et al., 2023). The research-
109
51 ers demonstrated that thaumatin elicits a sweet taste sensa-
110
healthier alternatives to traditional sugar-based sweeteners.
52 111
As concerns over the adverse health effects of excessive tion, often described as clean and well-rounded, making it a
53 112
sugar consumption have grown, researchers have turned to desirable alternative for enhancing the palatability of various
54 113
55 natural compounds that possess sweet tastes without the food and beverage products. Collectively, these recent stud- 114
56 caloric burden or metabolic drawbacks associated with sug- ies underscore the ongoing exploration of bioactive mole- 115
57 ars. Recent articles in the scientific literature highlight the cules as sweeteners, offering a glimpse into the potential of 116
58 117
59 Q1 CONTACT Rafael Trindade Maia rafael.rafatrin@gmail.com 118
ß 2023 Informa UK Limited, trading as Taylor & Francis Group
 2 R. T. MAIA ET AL.

119 these compounds to reshape the landscape of sugar alterna- 178

120 tives in the quest for healthier dietary options. 179
121 Miraculins (Mir) are glycoproteins from plant origin, pri- 180
122 marily isolated from the miracle fruit, scientific name 181
123 Richardella dulcifica, a shrub native to West Africa. Its ability 182
124 to modify flavour was associated with a miracle, because it is 183
125 responsible for the conversion of sour and bitter taste into 184
126 sweet, when miraculins meets the taste buds (Garcia et al., 185
127 2023; Misaka, 2013). 186
128 The search for natural compounds that have sweetening 187
129 properties, such as miraculin, is of great interest to society in 188
130 view to the relevant growth of chronic increasing diseases 189
131 such as diabetes, and the high consumption of simple sugars 190
132 is related to the increase in the disposition of metabolic syn- Figure 1. Miraculin homodimeric structure linked by a disulfide bridge. Source: 191
133 drome (Ronchillo & Joivin, 2019). There is a great demand in Adapted from Paladino et al. (2008). 192
134 the industry for better alternatives focused on the sweet- 193
135 eners production, or flavour modifying compounds, since the dimerization and prevalence of histidine residues (His30 and 194
136 synthetic sweeteners available in the current market, are His60) are crucial for protein functionality (Ohkura et al., 195
137 linked with the development of several types of cancers and 2018; Theerasilp & Kurihara, 1988). 196
138 degenerative diseases (Castiglia et al., 2018; Han et al., 2019). In the presence of acidic substance, miraculin can interact 197
139 Therefore, miraculin proteins have great potential for bio- with the palatability receptor cells located on the tongue, 198
140 technological applications in the food industry as sweeteners causing a chemical reaction, synapsing with the brain, and 199
141 or flavour enhancers in various products, which can be a causing a sensation of flavour modification, resulting in a 200
142 good associate with dietary prescriptions, in view of low-cal- sensation of strong sweet taste, masking acidic flavour and 201
143 orie concentrations and minimal glycaemic load (Garcia completely bitter flavour. Even after contact with acidic sub- 202
144 et al., 2023). However, miraculin production is scarce and its stances, its effect on sweet taste buds can last for about 1 to 203
145 purification is limited. 2 h (Igeta et al., 1991). 204
146 Therefore, to aim biotechnological applications for miracu- The mechanisms of interaction with the receptor 205
147 lins is necessary to design analogues (mimetic) peptides, cap- (T1R2/T1R3), is not yet fully known, studies show that mirac- 206
148 able to interact with the active site from human sweet taste ulin behaves as an agonist at acidic pH and inactive at neu- 207
149 receptor. In this review, potential applying of miraculin as tral pH, binding antagonistically to the receptor under these 208
150 sweeteners using protein-engineering techniques is conditions. Over the years, proteins homologous to miraculin 209
151 highlighted. has been discovered, isolated in several plants belonging to 210
152 the family Rutacea, Solanaceae, Rubiaceae. Based on 211
153 sequence and similarity, these proteins are called miraculin- 212
154 Miraculin protein like proteins (MLPs), when they exhibit 30 to 95% identity. 213
155 Some plants have ability to produce sweet-tasting com- Due to the amino acid sequence, MLPs are classified within 214
156 pounds, such as glycoproteins. Among them, stands out the family of soybean trypsin inhibitors of the Kunitz type 215
157 Miraculin (Synsepalum dulcificum), belonging to the (IST), exhibiting approximately 30% identity with IST 216
158 Sapotaceae family. The plant was recognized in 1968 in West (Cevallos-Mun ~oz et al., 2007; Ito et al., 2007). 217
159 Africa because of its red berry fruits (Campos & Gonzales, The orange tree species (Citrus sinensis) is included in the 218
160 2015; Ohkubo et al., 2015). Citrus genus, and in the biological family Rutacea. Among 219
161 The fruit has been studied since the 1980s by several the genes found naturally in this plant, there are miraculin 220
162 countries in the world and has different names or terms type genes, which were related to the potential bioinsecti- 221
163 such as Magic Berry, Miracle fruit, Miracle Berry, Miracle Fruit, cide capacity (Sanematsu et al., 2014; Takai et al., 2013). 222
164 223
Miraculous Berry, Sweet Berry and Miraculous Berry
165 224
(Ronchillo & Joivin, 2019).
166 The sweet taste in human food 225
Miraculins are characterized as glycoproteins from plant
167 226
origin or N-glycoproteins, belonging to the family of Kunitz The preference for sweet flavour has an innate and historical
168 227
trypsin-inhibiting proteins. The native miraculins has a origin, specialized taste cells begin to be developed in the
169 228
molecular weight of 42,000 Da, with a polypeptide chain intrauterine phase (approximately at 7–8 wk), they reach
170 229
structure containing about 191 residues, which have a flavor- maturity at around 13th weeks and already begin to transmit
171 230
172 modifying function when they are in homodimeric format, palatability information to the brain, the stimuli continue in 231
173 each monomer has N-glycosylated polypeptide chains, which breastfeeding and lasting for the rest of life (Mondego et al., 232
174 are interconnected by a disulfide bridge in asparagine resi- 2011; Sun et al., 2007). 233
175 dues (Asn42 and Asn186) and by interchain interactions of The sweet taste receptor, located in taste buds, is formed 234
176 cysteine residues, mainly CYS138 (Figure 1), which is directly by two types of receptors: T1R2 and T1R3, coupled to G pro- 235
177 linked to the other subunit. It is also known that the tein (GPCRs), and they are responsible for sending sweetness 236
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3

237 signals to the central nervous system. Five basic tastes are diseases, such as diabetes, cancer, coronary heart disease, 296
238 identified: bitter, sour, sweet, umami and salty. Historically, and even other comorbidities such as obesity; the search for 297
239 two tastes have shown greater relevance to the evolution of other alternatives to sweeten food has increased. The 298
240 species, bitter and sweet (Bachchu et al., 2011; Silva & Teles, demand for sweeteners from natural sources, such as miracu- 299
241 2013). lous ones only tend to grow, what led the biotechnological 300
242 Sweet taste was important for survival, as the availability community to develop products, or dietary sweeteners with 301
243 of substances rich in carbohydrates is associated to good miraculin, due to the high intensity, persistence, and preva- 302
244 sources of energy, and consequently energy storage, in add- lence of sweetness (Igeta et al., 1991; Shibao et al., 2009). 303
245 ition to familiarity, as well as greater exposure throughout In this sense, biotechnological use of miraculins can be 304
246 life, makes it preferable for the sweet taste. The bitter taste diverse, covering different areas of application, but mainly 305
247 was associated with foods with toxic components, existing in linked in the food and pharmaceutical industry areas. 306
248 the natural environment (Cul & Col, 2006; Ventura & Regarding food and flavour modifying capacity, miraculins 307
249 Mennella, 2011). had been used since the nineteenth century, to improve the 308
250 One of the first strategies to sweeten food was the use of palatability of acidic foods and sweeten beverages, by the 309
251 honey, used since ancient Greece and China, but with the native West African Indians (He et al., 2015; Mooradian, 310
252 development of the sugar industry, honey was replaced by 2019). 311
253 common sugar, sucrose. However, the overuse of sucrose The miraculous fruit application in commercial acidic 312
254 has relation to the increase of chronic diseases, such as obes- drinks, which contained in its composition great amount of 313
255 ity and diabetes. Which led society to seek new products citric acid, had better effects on the perception of sweet 314
256 and alternatives to replace sugar. Currently, the industry taste, compared to other types of acid, such as acetic acid, 315
257 offers several substances that can confer or intensify the demonstrated in the study (Gnanavel & Peddha, 2011). 316
258 sweet taste in foods; they are called sweeteners (Moreira, The application of the miracle fruit was performed in 317
259 2005; Ohtsu & Col, 2014). acidic drinks based on lemon, in relation to sensory charac- 318
260 Sweeteners are basically divided into two major groups: teristics, the results were like sucralose, which is one of the 319
261 nutritive sweeteners (NS) and non-nutritive sweeteners (NNS), most used sweeteners in the population. It provided a prom- 320
262 NS add caloric value to foods, within this group are sucrose, ising alternative as a substitute for sugar for a natural com- 321
263 lactose, fructose, and polyols, whereas NNS are sweet flavour ponent. What connoted in an indication for the potential use 322
264 components, which offer insignificant amounts or no calories as a flavour enhancer in Citrus-based drinks, or other prod- 323
265 in food, such as: cyclamate, saccharin, aspartame, stevia and ucts like soft drinks, where this type of acid is usually added 324
266 others. NNS, like dietary sweeteners, are food additive, which for the balance of flavour, odour, and quality maintenance 325
267 can totally or partially replace sugar in a range of products, (He et al., 2015). 326
268 and can be of high intensity, which are those that exert up to The pulp and seed of the miraculous fruits were intro- 327
269 a hundred times greater capacity to sweeten than sucrose and duced in the elaboration of a functional yogurt, the flavour 328
270 low intensity ones. They are generally used in combination modifying activity makes it possible to introduce sweet char- 329
271 with another type of sugar or compound (Chattopadhyay et al., acteristic foods, as well as savoury foods, such as cheeses, as 330
272 2014; Mahar & Duizer, 2007). well as in milk derivatives, acting as a flavour moderator 331
273 Sweeteners can also be classified according to their origin (Akinmoladun, 2016; Igarashi et al., 2013; Muller et al., 2018). 332
274 into: (i) natural, composed of organic substances found in Furthermore, a research (Freitas & Arau jo, 2010) describes 333
275 nature that cause sweetness, and (ii) artificial, which are syn- alternatives for replacing sugar with natural compounds 334
276 thetic substances, prepared in the laboratory, which are also including miraculin, influencing the manufacturing potential 335
277 responsible for to give sweet taste to foods (Weihrauch & for use as a sweetener, considering that the available syn- 336
278 Diehl, 2004). thetic sweeteners indicate having undesirable clinical effects.
337
279 The synthetic sweeteners most used by the industry are The miraculins property as an alternative sweetener and
338
280 sucralose, saccharin, aspartame, cyclamate and acesulfame. sugar substitute is commonly recognized and can be applied
339
281 However, due to side effects of long-term consumption, mainly to people affected by diabetes (Garcia et al., 2023).
340
282 linked to psychological problems, brain tumours, the devel- Once diabetes is a chronic disease, a change in eating
341
283 opment of glucose intolerance, papillae sensation loss, heart habits is necessary such as decreased consumption of carbo-
342
284 failure, and severe cancer has led to a great concern of use 343
hydrates and restriction to the use of refined sugar directed
285 in consumers and health professionals. That is why natural
344
the individual to the use of sweeteners. The use of miraculin
286 compounds or derivatives are of great interest for industrial
345
would be of great value, since its effect is not linked to
287 applications in the sweeteners manufacture, such as for
346
sweet molecules, but rather to the sensation of the sweet
288 example miraculin, which has a natural sweet feeling prop-
347
taste, when the active compound, miraculin binds with sweet
289 erty (Misaka, 2013; Ronchillo & Joivin, 2019; Yang, 2010).
348
290 taste receptors (Smith et al., 2022; Igarashi et al., 2013). 349
291 The safety of new proteins use was evaluated, where it 350
292 Biotechnological applications of miraculin
was demonstrated by means of proteomic analysis, and by 351
293 in vitro digestibility assay, that miraculin is quickly and com- 352
294 Due to the high sugar consumption and its probable contri- pletely digested by pepsin, which indicates that these pro- 353
295 bution to the appearance of chronic non-communicable teins may not be absorbed, reducing the risk of toxicity, and 354
 4 R. T. MAIA ET AL.

355 the release of insulin by the pancreas (Fazilah et al., 2019). In modification, which would be an obstacle for mass produc- 414
356 addition, they also analysed in silico the allergenic or toxi- tion. In addition, the original miraculin molecule has a 415
357 genic effect of Mir, showing that it did not represent such molecular weight around 90 KDa, which makes production in 416
358 risks. Miraculin can also be used in the pharmaceutical indus- the laboratory more difficult and more expensive (Agblekpe 417
359 try to increase the palatability of medicines, such as syrups et al., 2016; Ohkura et al., 2018). Therefore, the production of 418
360 (Chattopadhyay et al., 2014). functional peptides analogous to miraculin would be of great 419
361 use for biotechnological production. 420
362 Industrial production limitations 421
363 Computational tests for selection and rational design of 422
364 The production of miraculin by means of the native plant on
industrial molecules 423
365 a large scale is limited, considering that large plantations 424
366 would be necessary to obtain small amounts of purified The advancement and improvement in the computers data 425
367 miraculin, in addition to which, for the protein displays a processing capacity, enabled the bioinformatics develop- 426
368 potential flavour-modifying effect, it must present structural ment, where the use of computational tools is focused on 427
369 conformation suitable to bind to the sweet taste receptors, the study of biological systems, and possible applications for 428
370 T1R2 and T1R3. In addition, to being a high molecular problems related to human health, such as molecule design 429
371 weight glycoprotein, its production in the original format for drugs or bioactive substances (Tafazoli et al., 2019). 430
372 would have great costs (Cevallos-Mun ~oz et al., 2007; The study of bioinformatics aimed at rational design, can 431
373 Holloway et al., 1996; Shibao et al., 2009). be applied both in the discovery of potential substances, for 432
374 Regarding cultivation, the Synsepalum dulcificum plant has the preparation of drugs, but also, it can be used to maxi- 433
375 a prolonged youthful period and slow growth rate, which mize the uniformity and functionality of existing substances, 434
376 hinders industrial production. Furthermore, it is an endan- both obtained by the rational design itself, as well as also of 435
377 gered species, seen as likely recalcitrant species, that is, the substances found naturally. In this way, the methods of 436
378 seeds drastically decrease viability when air-dried, little is development of bioactive compounds have moved from the 437
379 known about the storage and conservation of the germina- laboratory to computational studies with greater intensity, 438
380 tive activity of the seeds, which hinders conservation, storage because the activity models generated have been showing 439
381 and planting (Agblekpe et al., 2016; Holloway et al., 1996). efficiency and fidelity in the prediction of biological target 440
382 Miraculins extracting and purifying methods from molecules (Matsuyama et al., 2009; Tchokponhoue et al., 441
383 Synsepalum dulcificum were analysed (Shibao et al., 2009). 2019). 442
384 For the extraction of total protein, some buffers were com- By providing rationality, the techniques used can previ- 443
385 pared (phosphate buffer, saline, NaCl and tris-HCL), and for ously rank the essential components and delete the inactive 444
386 the protein purification, used immobilized metal ion affinity ones. In addition to that, the computational techniques can 445
387 chromatography (IMAC) with nickel-NTA, and demonstrated explore faster many sequences, simplifying the process of 446
388 that this method of purification in a single step process is identification and selection of molecules, connoting improve- 447
389 satisfactory, however the total volume of purified miraculin ment in efficiency and research performance, reducing time 448
390 was still unsatisfactory. and cost with experimental methods. After demonstrating 449
391 What becomes an impasse for industrial production, in high selectivity and affinity with the molecule of interest, 450
392 addition to the miraculous fruit, outside the native environ- studies should go in partnership with experimental (in vitro 451
393 ment is difficult to cultivate. In this way, some studies have and in vivo) techniques (Ezura & Hiwasa-Tanase, 2016; 452
394 moved towards the heterologous production of proteins like Matsuyama et al., 2009; Tchokponhoue et al., 2017). 453
395 native miraculin, both artificially inserting the gene in organ-
454
396 isms such as bacteria, yeasts, however the recombinant Mir
455
397 expressed through these transgenic organisms has not
Rational peptide design 456
398 shown satisfactory results in inducing the modifying activity
457
For the rational design of peptides, there are four types of
399 of flavour (Agblekpe et al., 2016; Holloway et al., 1996). The
458
methodological procedures most used, which are the (i)
400 459
expression is also found in plants with easier cultivation, methods based on a template sequence, (ii) physicochemical
401 460
such as tomatoes (Solanum lycopersicum), lettuce (Lactuca methods, (iii) evolutionary methods or (iv) de novo methods
402 461
sativa) and grapes (Vitis vinifera), to express and obtain (Muttenthaler et al., 2021).
403 462
greater amounts of purified miraculin (Leal et al., 2019). A Methods based on a template sequence is usually chosen
404 463
study (Ohkura et al., 2018) was developed of heterologous when there are studies on a known sequence, which
405 464
production of miraculin in grapes (Vitis vinifera), where it pre- presents considerable activity, but which still needs to be
406 465
407 sented a homology of 61%, suggesting that this recombinant improved, and can be in the perspective of size reduction, 466
408 miraculin could have flavour-modifying activity, he also eval- increased selectivity, greater stability, reduction, or inhibition 467
409 uated and confirmed that protein produced managed to cytotoxic effects. The construction takes place through the 468
410 demonstrate inhibitory activity against trypsin. identification of the key amino acids (aa), replacement of aa 469
411 However, its production by heterologous expression is still residues and deletion of aa that has no crucial activity for 470
412 economically expensive and needs to be in the proper con- the stability and action of the molecule (Pearce et al., 2019). 471
413 formation as a homodimer to trigger the flavour In an interesting study (Zhang & Skolnick, 2004), it was 472
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

473 designed a peptide rich in alanine (Pa-MAP2), based on a 532

474 peptide synthetically produced and obtained from fish 533
475 P. americana, the substitutions of aa led to greater specificity 534
476 and less toxicity for mammals. 535
477 The physical-chemical methods are based on the mechan- 536
478 ism of action, this method is generally chosen for peptides 537
479 that have a-helix conformation, as this method easily analy- 538
480 ses a spectrum of activities and properties, such as total 539
481 charge, hydrophobicity, generally its information is coupled 540
482 to the method based on model sequence or other rational 541
483 design methods (Muttenthaler et al., 2021). 542
484 The two methods that need a high computational power 543
485 are the evolutionary methods and de novo methods. For the 544
486 design through evolutionary forms, it is necessary the aid of 545
487 genetic algorithms, which analyse a group of sequences, 546
488 deletion of aa in the evolution and similarity. The methods 547
489 again, use aa patterns or frequency, and positioning prefer- 548
490 ences, to generate a variety of analogous sequences 549
491 (Arodola & Soliman, 2017; Muttenthaler et al., 2021). 550
492 EvoDesign is an online server (Bazzoli et al., 2011; Menetti, 551
493 2006), which uses evolution-based profiles to direct the 552
494 search simulation sequence; it searches for complete atomic 553
495 models of a PDB scaffold, produces a list of projected 554
496 sequences and indicates the identity percentage of the 555
497 sequences generated based on the scaffold structure. 556
498 Scaffold is the structure of the specific chain of a complex, 557
499 which serves to increase affinity, fixation with another com- 558
500 plex or binding partner and improve stability, since most Figure 2. Overview of EvoDesing server strategies. Source: Pearce et al. (2019). 559
501 protein functions are mediated by protein-protein interac- 560
502 tions. The scaffold is part of the interface design protocol, The high-scale design, with foldable molecules, indicates that 561
503 the protocol can be applied for several purposes, one of the the combination of evolutionary profiles with the knowledge 562
504 applications is the design of bioactive proteins, which can be of physical energy, is more satisfactory than the protocols 563
505 redesigned to increase the affinity to a fixed receptor, this that are based only on physics (Costa, 2018; Holloway et al., 564
506 methodology showed satisfactory results to the redesign of 1996). 565
507 various protein systems. This indicates a great potential for 566
508 the synthesis of new therapeutic molecules, enzymes and 567
509 other active compounds (Fjell et al., 2011).
Molecular modelling 568
510 For the user to perceive the quality of the generated Due to limitations of experimental techniques, computational
569
511 sequences, the server also provides information on solvent tools have been increasingly developed, with constant
570
512 accessibility, torsion angle of the backbone and possible rela- improvement of the base algorithms used, using methodolo-
571
513 tive errors normalized for the projected secondary structure. gies based on physical equations and theories and computer
572
514 In addition to the server being based on evolutionary pro- skills. According to this perspective studies of molecular
573
515 files, the method shows an advantage for coupling know- modeling, molecular dynamics and molecular docking are
574
516 ledge based on physics. After the structured profiles are
575
crucial to a better understanding of molecules functions and
517 produced from the protein fold and protein interfaces, the
576
applications. Molecular modelling, for example, analyses and
518 physical-chemical chain and the main chain are accommo-
577
predicts the three-dimensional (3D) structure of a protein by
519 dated through a force field. The final sequences produced
578
simulations, enabling important information about the loca-
520 579
are organized through a grouping by an algorithm of all tion of aa, through the development of computational codes
521 580
sequence decoys designed during the Monte Carlo (MC) (Muttenthaler et al., 2021; Porto et al., 2012).
522 581
simulation of replica exchange (Arodola & Soliman, 2017; Computational modelling methods have been a good
523 582
Fjell et al., 2011). alternative for the study of protein structures, as they are
524 583
EvoDesign server features two design options, “Monomer cheaper than the approaches of X-ray crystallography and
525 584
Design”, which uses monomeric proteins, and features
526 NMR (Magnetic Resonance) spectroscopy; in addition to that, 585
527 “Interface Design”, which can work with more complex, pro- in computational models it is possible to predict results 586
528 tein-protein interaction structures. Both options are worked experimental models. Modelling can be done using methods 587
529 in three stages, the pre-processing, simulation, and analysis that do not depend on a template free template structure – 588
530 of the data produced during the simulation, which will prediction ab initio and again, and those that do are tem- 589
531 result in the final projection of the sequences (Figure 2). plate based – comparative modelling and threading. 590
 6 R. T. MAIA ET AL.

591 The ab initio (template free) methods are based on the changing the shape, this model is applied only in protein-lig- 650
592 knowledge of thermodynamics, which seeks to find the min- and docking, mainly in the design and development of drugs 651
593 imum global free energy, corresponding to the energies of (Muttenthaler et al., 2021). 652
594 native protein structures. As for methods based on compara- In rigid docking, the molecular surfaces of both structures 653
595 tive modelling, or model based on homology (template are considered as rigid in their entirety; only the rotational 654
596 based), it is based on the use of a sequence of template and translational degrees of freedom of the ligand are con- 655
597 amino acids, which is aligned with the aa sequence that one sidered, being fixed in a rigid structure. It is understood that, 656
598 aims to project, it is one of the most used methods to study even if the surfaces are rigid, one of the proteins in the com- 657
599 the structure of proteins (Ahn et al., 2006; Migliolo et al., plex will penetrate the other (Hung & Chen, 2014). 658
600 2016; Mitra et al., 2013). There are other theories and variation of the rigid dock- 659
601 Threading modelling is also a methodology (template ing, called soft flexible docking, where variations in the sur- 660
602 based), where a sequence of aa considered as a template is face of both structures (receptor/ligand) are allowed, 661
603 crossed with the data from a topology bank, resulting in a provided they are between a surface atom and a central 662
604 new structure. If there is an evolutionary relationship, it is atom or both surface atoms (Muttenthaler et al., 2021). 663
605 determined that the modelling is by homology, if there are 664
606 no structural characteristics of the evolutionary relationship,
Ab initio methods
665
607 the modelling should be by threading (Ahn et al., 2006; 666
608 Mitra et al., 2013). The ab initio methods are very precise and consistent 667
609 because they are semi-empirical, they are based only on the 668
610 laws of quantum mechanics, electron masses and charges, 669
611 Molecular docking 670
atomic nuclei, and fundamental physics constants, speed of
612 The term docking is used to check a set of computational light and constant of Plank, used in computational chemistry. 671
613 techniques that try to determine, through atomic coordi- From Latin, the expression means “from the beginning”. The 672
614 nates, the best association or fit between two structures, methods are based on two principles: the first is based on 673
615 being normally applied to a receptor and a ligand (Dorn the theory that the information of the amino acid sequence 674
616 et al., 2014; Pearce et al., 2019). is responsible for producing the three-dimensional structure 675
617 In summary, docking perceives the connection mode by of the protein, occurring an envelope of structures to a 676
618 the orientation and conformation of a small molecule that is native state, which is, found with the minimum of global 677
619 the ligand, with the connection site of a large molecule that energy (Sliwoski et al., 2014; Yunta, 2016). 678
620 is the receptor, allowing the coupling and formation of a sta- These methods are intended to solve the Schro €dinger 679
621 ble complex (Dorn, 2008; Halperin et al., 2002; Pearce et al., equations for the system, resulting in energy and wave func- 680
622 2019). tion data. However, this equation is not well applied to mol- 681
623 The connection affinity of the complex can be measured ecules with more than one electron, so, for simulations with 682
624 using punctuation functions; the layout of the molecular larger systems, it is considered a harmonic approximation 683
625 docking is shown in. The ability to complex a molecule with system to facilitate calculations (Muttenthaler et al., 2021; 684
626 another target structure depends on the ability to interact Yunta, 2016). 685
627 positively with a specific binding site, thus, the structures With the development of methods that use approxima- 686
628 that share these positive interactions, are probably capable tions, such as those based on electronic density, Density 687
629 of exerting active biological effects (Santoyo et al., 2013). Functional Theory (DFT), Local Density Approximation (LDA) 688
630 What makes protein-ligand or protein-protein binding sta- and Generalized Gradient Approximation (GGA), associated 689
631 ble are intermolecular or non-covalent interactions as inter- with the improvement of computational power in recent 690
632 actions of hydrogen bonds, electrostatic, hydrophobic, and times, the analysis of complex biological systems has been 691
633 other interactions. A range of possibilities for available dock- showing satisfactory results, with a high processing rate 692
634 ing software is available, the most used ones are AutoDock, when performing the calculations, and low computational 693
635 DOCK, LUDI, Glide, FlexX, GOLD and AutoDock Vina (Dorn, cost (Matta, 2010; Vakser, 2014). 694
636 695
2008; Grinter & Zou, 2014).
637 696
An important factor in choosing the possibilities is the
638 Molecular fractionation with conjugated caps (MFCC) 697
protein-mooring model. The approaches related to rigidity
639 698
are divided into three, flexible docking, semi-flexible docking, To enhance the studies of biological systems with many
640 699
and rigid docking. Flexible docking is when the receptor and atoms, the Molecular Fractionation with Conjugated Caps
641 700
the ligand are adaptable and flexible bodies, analysed by the (MFCC) method was developed, which provides results at the
642 701
degrees of freedom of both molecules. Semi-flexible docking
643 ab initio level of interaction energies between a protein and 702
644 is when one element of the complex is considered rigid, and another structure. Its results are based on the division of the 703
645 the other is not. The algorithms generally consider that the three-dimensional molecular structure, in the groups of 704
646 molecular surface of the ligand is flexible, where all degrees amino acids, thus demonstrating, as a result, the energy gen- 705
647 of freedom of the ligand, rotational, translational, and con- erated between the protein and the ligand through the com- 706
648 formational are considered, due to being a protein sequence binations between the small fragments or groups analysed 707
649 with smaller dimensions, with greater probabilities of (Ewars, 2011; Matta, 2010). 708
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7

709 768
710 769
711 770
712 771
713 772
714 773
715 774
716 775
717 776
718 777
719 778
720 779
721 780
722 Figure 3. Schematic representation of the MFCC method. 781
723 782
724 This technique is based on the thermochemical theory. of the ligand with the caps is denoted, and finally, the fourth 783
725 The energetic description of a given microsystem is calcu- term (T4) corresponds to only caps energy. 784
726 lated from Schro €dinger’s Hamiltonian equation, which con- 785
727 sists in the fragmentation of a three-dimensional structure 786
728 (macromolecules) into smaller individual fragments. Based on
Final considerations 787
729 amino acids that presented a close interaction, this strategy Glycoproteins such as miraculins are complex biomolecules 788
730 has the purpose of conserving the orbital properties of the that perform essential biological functions. The size of mirac- 789
731 analysed residues. The neighbouring amino acids (caps) and ulins is approximately 190 amino acids, which hinders their 790
732 their respective peptide bonds serves to simulate and not industrial production. This opens a gap for studies that focus 791
733 break the valences that would naturally occur in the com- on the rational design of mimetic peptides, that is, smaller
792
734 plete system or protein (Gordon et al., 2012; Vakser, 2014). peptides that simulate the action of these proteins.
793
735 The calculation of the interaction energy E (L - Ri) is The use of computational techniques for protein engin-
794
736 eering allowed incredible advances in almost all related
795
expressed in kcal mol-1, between a ligand L and an amino
737 796
acid residue Ri of the protein, being defined by the equa- areas. In the specific case of miraculins as sweeteners, pro-
738 797
tion: tein engineering is an incredible opportunity to generate
739 798
useful products, with commercial demand, with benefit and
740 EðL − RiÞ ¼ EðL − Ci − 1RiCi þ 1Þ − EðCi − 1RiCi þ 1Þ 799
741 safe for human consumption. In the future, where sweet- 800
742 − EðL − Ci þ 1Þ þ EðCi − 1Ci þ 1Þ eners possibly will be produced and consumed in the order 801
743 When ligand L is aa residue (L ¼ Rj) from another protein,
of consumption of certain commodities, it is extremely inter- 802
744 it binds to aa from the receptor, the equation can be given,
esting that they use emerging techniques to develop better 803
745 as follows:
products. The computational design of proteins represents, 804
746 in this sense, an innovative, fast, safe, and incredible poten- 805
747 EðRj − RiÞ ¼ EðCj − 1RjCj þ 1 − Ci − 1RiCi þ 1Þ tial technique to be applied in the food field. More research 806
748 −EðCJ − 1Cj þ 1 − Ci − 1RiCi þ 1Þ is needed for this new field of knowledge. Q2 807
−EðCj − 1RjCj þ 1 − Ci − 1Ci þ 1Þ
749 þEðCj − 1Cj þ 1 − Ci þ 1Þ
808
750 809
Acknowledgments
751 where E (Cj − 1RjCj þ 1 - Ci − 1RiCi þ 1) corresponds to the 810
752 total energy of the system produced by the two residues We acknowledge for CAPES (Coordination for the Improvement of 811
753 (ligand and receptor), that interact mutually together with Higher Education Personnel) for the fellowship granted for the first and 812
754 the hoods; the term E (Cj − 1Cj þ 1 - Ci − 1RiCi þ 1) is equiva-
second authors. 813
755 lent to the energy of the residue Ri with its conjugated 814
756 hoods Ci − 1Ci þ 1 and Cj − 1Cj þ 1 of Rj. Corresponding to Disclosure statement 815
757 E (Cj − 1RjCj þ 1 - Ci − 1Ci þ 1) there is the energy system 816
758 formed by Rj and other hoods. The fourth term E (Cj − 1Cj þ
No potential conflict of interest was reported by the author(s). 817
759 1 - Ci − 1Ci þ 1) suggests the energy of the fragment formed
818
760 only by the de and Rj hoods (Mota, 2016). The simplified lay- Funding 819
761 820
out of the MFCC method is shown in Figure 3:
762 The author(s) reported there is no funding associated with the work fea- 821
The term (T1) da refers to the total energy of the system,
763 tured in this article. Q7 822
formed by the residue of the ligand and residue of the
764 823
receiver with its caps. In the second term (T2) the total
765 ORCID 824
766 energy of the waste of the receiver with its caps is calcu- 825
767 lated, in the third term (T3) the total energy of the residue Rafael Trindade Maia http://orcid.org/0000-0002-5870-1091 826
 8 R. T. MAIA ET AL.

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