Question

Mirror-Image Proteins As noted in $\underset{―}{\text{ Chapter }3}$, "The amino acid residues in protein molecules are almost all L stereoisomers." It is not clear whether this selectivity is necessary for proper protein function or is an accident of evolution. To explore this question, Milton and colleagues (1992) published a study of an enzyme made entirely of $\mathrm{D}$ stereoisomers. The enzyme they chose was HIV protease, a proteolytic enzyme made by HIV that converts inactive viral preproteins to their active forms. Previously, Wlodawer and coworkers (1989) had reported the complete chemical synthesis of HIV protease from L-amino acids (the L-enzyme), using the process shown in Eigure 3-30. Normal HIV protease contains two Cys residues, at positions 67 and $95.$ Because chemical synthesis of proteins containing Cys is technically difficult, Wlodawer and colleagues substituted the synthetic amino acid L- $a$-amino- $n$-butyric acid (Aba) for the two Cys residues in the protein. In the authors' words, this was done to "reduce synthetic difficulties associated with Cys deprotection and ease product handling." a. The structure of Aba is shown below. Why was this a suitable substitution for a Cys residue? Under what circumstances would it not be suitable?

Short answer

Aba is a suitable substitution for Cys due to similar size and chemical simplicity, avoiding oxidation or disulfide bond complications unless disulfide bonds are crucial for functionality.

Step-by-step solution

Understand the Chemical Structure of Cys and Aba

Cysteine (Cys) is a sulfur-containing amino acid characterized by its side chain with a thiol group (-SH), which is crucial for forming disulfide bonds in proteins. L-α-amino-n-butyric acid (Aba) is a non-natural amino acid whose structure does not include sulfur or the ability to form disulfide bonds.

Analyze the Chemical Properties

The substitution from Cys to Aba might be chemically suitable due to its similar size and chemical structure, lacking a reactive side chain like the thiol group. This substitution can reduce complexities related to handling Cys, such as its propensity to form disulfide bonds or undergo oxidation. Changes in these characteristics are often required to ensure easier synthesis and handling of proteins.

Evaluate Suitability of Substitution

Replacing Cys with Aba is suitable if the protein does not require disulfide bond formation for structural integrity or function. Therefore, this substitution is suitable in environments where disulfide bonds are non-essential, and Cys's reactive thiol group could lead to unwanted chemical reactions during synthesis.

Identify When the Substitution is Not Suitable

This substitution would not be suitable if disulfide bond formation is critical for the protein's stability, structure, or activity. For proteins that rely on disulfide bonds to maintain their tertiary structure or catalytic activity, replacing Cys with Aba could impair their function.

Key concepts

Amino Acids

Amino acids are the building blocks of proteins. They are organic compounds made up of an amino group ${\text{-NH}}_2$, a carboxyl group $\text{-COOH}$, and a distinctive side chain (R group). This side chain differentiates one amino acid from another. There are 20 standard amino acids that are encoded by the genetic code. Each of these is involved in protein synthesis. Amino acids exist as stereoisomers, known as L and D forms. Proteins in living organisms are made up almost exclusively of L-amino acids.

• L-Stereoisomers: Commonly found in proteins synthesized by living organisms.
• D-Stereoisomers: Rarely found in nature, except in some bacterial cell walls.

The selection of L over D stereoisomers may have originated early in evolution. However, certain studies, like the one by Milton and colleagues, explore the viability of proteins made entirely from D-forms, such as the case with HIV protease.

HIV Protease

HIV protease is an enzyme crucial to the life cycle of the Human Immunodeficiency Virus (HIV). This proteolytic enzyme divides long inactive protein chains into smaller, active fragments. These fragments are necessary for the virus to mature and become infectious. The key features of HIV protease include:

• It plays a role in refining viral proteins, making it vital for HIV replication.
• Its structure consists of two identical subunits, which together create an active site where protein cleavage occurs.
• The enzyme has been synthesized both with natural L-amino acids and entirely synthetic D-stereoisomers for experimental purposes.

HIV protease contains crucial amino acids like Cysteine (Cys), which can form disulfide bonds, influencing the enzyme's structure and resilience.

Protein Synthesis

Protein synthesis is the process every cell uses to build proteins. It uses two main stages: transcription and translation. During transcription, DNA is used to create messenger RNA (mRNA) in the nucleus. Then, during translation in the cytoplasm, ribosomes read mRNA sequences to build the corresponding protein from amino acids. Every protein is synthesized starting from the N-terminal (amino end) and ending at the C-terminal (carboxyl end). The sequence of amino acids in a protein determines its structure and function. Protein synthesis is a precise process that, in the case of developing HIV protease, has been manipulated to include either natural amino acids or synthetic alternatives to explore structural variations and functionalities of proteins.

Disulfide Bonds

Disulfide bonds are covalent bonds that form between two sulfur atoms, typically from the side chains of two Cysteine residues. These bonds play a vital role in stabilizing the three-dimensional structure of proteins. Disulfide bonds contribute to:

• Maintaining protein integrity by linking different parts of the protein chain.
• Stabilizing protein foldings against conditions like heat or pH changes.
• Alternating structural properties to enable protein functionalities.

In the synthesis of proteins like HIV protease, disulfide bonds, facilitated by Cys residues, are essential unless alternative synthesis, such as substitution with Aba, is employed due to the practical challenges of handling reactive thiol groups. While Aba cannot form disulfide bonds, it can be suitable in moves to simplify synthesis where such bonds are non-essential.