Protein Functionalization

 Proteins are the engines of a living cell. Within and around cells they perform a magnificently diverse set of functions. They do most of the work in cells and are required for the structure, function, and regulation of the body’s tissues and organs. Besides providing structure and stability, proteins are involved in cell signalling, catalysing reactions, storage & transport, and are therefore extensively studied. Over the years, tools have become available for researchers to reveal structure and function relationships, as well as localization and their interactions with other proteins.

Nature routinely causes modifications of proteins in specific sites, enabling a dramatic increase in functional diversity.  The evolution of living beings is based on this functional diversity. The increase in research and technologies are leading humans to a state wherein it is possible to manipulate and add functional properties to specific proteins. However, modifying such chemically and structurally rich biopolymers without perturbing their function remains an open challenge.

1. Selective Chemical Protein Modification

Chemical modification of proteins is an important tool for probing natural systems, creating therapeutic conjugates, and generating novel protein constructs. Site-selective reactions require exquisite control over both chemical and regioselectivity (regioselectivity occurs in chemical reactions where one reaction site is preferred over another), under ambient, aqueous conditions. There are now various methods for achieving selective modification of both natural and unnatural amino acids—each with merits and limitations.

Potential transformations, if they are to be relevant, are moulded by the need for biologically ambient conditions (that is, <37 °C, pH 6–8, aqueous solvent) so as not to disrupt protein architecture and/or function. Ideally, this should proceed with the near-total conversion to generate homogenous constructs.

2. Constrains of Protein Functionalization

While many past examples of so-called ‘bioconjugation’ exist, those that teach a strong strategic lesson are rarer. The rigour of the chemical approach (including proper characterization) has been lacking—supplanted perhaps by a pragmatic desire for a useful product. In an era now hungry for precise molecular knowledge of protein function, historical examples of precise protein chemistry become vital.

Although many strategies exist to label proteins specifically, these random modifications can adversely block enzymatic active sites and binding pockets or alter the protein’s 3D structure, leading to a decrease in or complete destruction or masking of activity. Despite these challenges, early pioneering work utilizing specific installation of functional groups onto antibodies has revolutionized biology, enabling the foundational advances making up immunohistochemistry and enzyme-linked immunosorbent assays (ELISAs).

3. Strategies of Functionalization

Regardless of the functionalization strategy, careful consideration must be paid to the reaction conditions for protein modification. Due to the fragility and massive size of most full-length proteins, the chemistries employed must occur under mild, aqueous conditions and proceed relatively rapidly on substrates at very low-molar concentrations. Owing to the diversity and arrangement of amino acid residues within each species, the protein targets display a diverse array of chemical functionalities with varying physicochemical parameters.

Targeting a specific functional group from the many that may be available on the protein’s surface, let alone a single moiety on a specific residue among many near-identical copies, is a tall order. To increase the specificity of these labelling reactions, three broad strategies have emerged:

1. Utilize reactive small-molecule chemical reagents to modify endogenous proteins.
2. Harness or hijack protein translational processes for direct labelling.
3. Utilize enzymes for co- or post-translational modification of proteins.

Small-molecule-based labelling strategies exploit differences in physicochemical characteristics (redox potential, nucleophilicity, acid dissociation constant, etc.) of a given sidechain to gain chemo selectivity.

4. Protein Functionalization in Food 

During extraction and food processing, proteins can be modified in multiple ways. Physical modifications (for example thermal treatment) and chemical modifications (for example ingredient interactions) can not only influence each other but also significantly affect the structural properties and functionality of proteins.

To obtain pure proteins or to design food products that contain (added) proteins, it is necessary to separate the desired protein from unwanted proteins and non-protein components present in the starting material. With variations that depend on the protein raw material, the process of producing bifunctional protein hydrolysates and peptides from food proteins typically involves extracting crude proteins from the protein source using aqueous or organic solvents and centrifuging the extract to further purify and separate the isolated proteins from unwanted and often insoluble non-protein materials.

Further purification steps may include dialyzing the supernatant of the protein extract against distilled water to remove residual salt and precipitate salt-soluble contaminants or treating the extract with dilute acid to initiate precipitation of the protein of interest (or that of the impure sediment) while leaving the impurities (or the desired protein) in solution. The proteins obtained from this kind of extraction and precipitation process are referred to as protein isolates and concentrates and could undergo additional purification based on their size, affinities for certain ligands, hydrophobicity, and ionic properties to obtain a purer, more homogeneous protein product.

5. Sources of Food Proteins
   • Conventional
     • Plants: Food proteins and their component bioactive peptides have been isolated from a variety of plant foods. The wide distribution and heterogeneity of the plant protein sources not only demonstrate the structural diversity, abundance, and diverse origins of plant food proteins but also the enormous potential for isolating novel peptides with various important bioactive properties from these plant protein sources.
     • Animals: Animal proteins are an important and often essential component of various food products where their physicochemical and biological characteristics serve to enhance the nutritional, organoleptic, and even health-promoting properties of those foods.

The biological properties of protein hydrolysates and peptides of animal origin have also contributed to their use in the food industry for the formulation of medical foods designed to manage food allergies and control conditions such as cystic fibrosis, liver disease, Crohn’s disease, and phenylketonuria. Bioactive hydrolysates and peptides have been derived from a myriad of diverse animal and marine protein sources and protein by-products including salmon, oyster, milk, eggs, etc.

• Novel
  • Insects: Although the heightened demand for high-quality food proteins and growing food security concerns in recent years have contributed to the increased use of proteins from insects for both food and feed, the consumption of insects, or entomophagy, is hardly a novel idea given that insects were a part of the diet of the evolutionary precursors of humans.

It is estimated that up to 2000 different insect species are edible and could be consumed at different stages of development, such as egg, larva, or pupa, with some of the most popular including locusts, crickets, caterpillars, bees, wasps, and ants. Insects are relatively rich in high-quality proteins with an essential amino acid content of 46–96%.

• Algae: Factors contributing to the growing use of algae include their relative ease of cultivation even on non-arable lands, high sunlight utilization efficiency and capacity to be grown using seawater and on residual nutrients. Apart from their protein content, algae are known to contain substantial amounts of other nutrients such as B vitamins and polyunsaturated fatty acids. Proteins from algal sources have also been used to produce health-promoting bioactive peptides.

The growing demand for protein foods presents both opportunities and challenges for researchers and food product developers. For instance, in seeking to produce sustainable, more affordable but also nutritious protein-rich foods from insects, scientists must also confront the microbiological, chemical, physical, and allergenic risks inherent in using members of the class Insecta for food.

Although the use of emerging green technology for food processing continues to enjoy growing popularity, there is a need to increase efforts towards scaling up reported beneficial results. It is also important to understand the mechanisms by which the alteration of protein structure results in functional changes.

6. References 

1. https://www.nature.com/articles/ncomms5740
2. https://gtr.ukri.org/projects?ref=EP%2FS033912%2F1
3. https://www.nature.com/articles/s41586-018-0608-y
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995230/
5. https://www.redalyc.org/journal/1698/169856422001/html/
6. https://www.sciencedirect.com/science/article/pii/S2590238519303479
7. https://www.wur.nl/en/Research-Results/Chair-groups/Agrotechnology-and-Food-Sciences/Food-Process-Engineering/Protein-functionalization.htm