Modular design of synthetic protein mimics, construction of helices

Abstract

Proteins are essential components of all living cells, and they play multifaceted roles in the vastly complex cellular assemblies within an organism. They control several vital biological processes and display a remarkable degree of specificity in their biological interactions. The widespread interest in understanding the structure and function of these molecules underscores their immense importance. The design of synthetic molecules with protein like properties is currently an area of intense activity. Such designed proteins can help clarify important questions regarding structural organization, stability, and function. Additionally, these design strategies may aim to generate protein mimics with tailor made properties such as thermal stability, solvent compatibility, and catalytic activity. This thesis is concerned with the synthesis and structural analysis of peptides designed to adopt specific secondary structures, as part of a program to develop methods for de novo protein design. The strategy is to develop stereochemically rigid synthetic model peptides that adopt helical or extended strand conformations, resembling secondary structural elements found in natural proteins. These “modules” can then be assembled, using linking residues or peptide sequences, to generate a synthetic mini protein incorporating more than one secondary structural element. Central to this strategy is the use of stereochemically constrained non protein amino acids along with selected protein amino acids to direct the folding of the polypeptide chain in the desired manner.

This thesis consists of eleven chapters. Chapter 1 - Introduction A general introduction is presented, covering:

various approaches to mimicking structural and functional properties of proteins, chemical modification and site directed mutagenesis used to investigate structure–function relationships, approaches to de novo protein design, a brief review of protein structural organization and protein folding, relevant to the present investigation.

Chapter 2 - Rationale and Design Principles This chapter details the rationale behind the design approach, emphasizing amino acids with strong stereochemical preferences for specific regions of ( / ) conformational space. The amino acids discussed include:

, dialkylated amino acids (Aib, Deg, Dpg), 1 aminocycloalkane 1 carboxylic acids, , unsaturated amino acids, proline and pipecolic acid, disulfide linked cystine residues.

General guidelines for using these residues to direct chain folding are provided.

Helix Design Using Aib (Chapters 3–7) Helices account for ~25% of secondary structures in proteins and are energetically stable units. The major emphasis of this work is Aib based helix construction, as Aib strongly promotes helicity due to steric constraints. The studies aim to determine:

the minimum number of Aib residues required to stabilize a helix, the tolerance for incorporating other amino acids without disrupting helix stability.

Solution conformations were studied using:

NMR, circular dichroism, temperature coefficients, solvent accessibility, 2D NOE experiments (to impose distance constraints).

Several peptides yielded single crystals, enabling high resolution X ray structure determination.

Chapter 3 - Peptides Boc Aib-(Ala Leu Aib) OMe and Boc-(Ala Leu Aib) OMe All four peptides adopt helical conformations in CDCl and MeOH. ROESY experiments confirm complete helicity. X ray structures reveal:

helical conformations in the solid state, intriguing features such as water insertion and apolar peptides acquiring amphiphilic character, particularly in hexapeptides and decapeptides.

Chapter 4 - Isomeric Decapeptides Two decapeptides isomeric to Boc Aib-(Ala Leu Aib) OMe were synthesized. Both peptides are helical in CDCl /MeOH, with slight terminal destabilization in DMSO. Comparisons show that Aib positioning causes only minor conformational differences. Crystal structures suggest packing dominates solid state structure over sequence specifics.

Chapter 5 - Peptides Incorporating Valine Val is typically considered a strand promoting residue, yet peptides containing Val–Ala–Leu–Aib repeats (8–16 residues) still adopt predominantly helical conformations. Helicity persists even with only 25–33% Aib content. Chain length dependent solubility effects were highlighted:

long peptides = poorly soluble in DMSO, N acetylation reduces solubility in chloroform but enhances helicity in DMSO.

All peptides showed helical conformations in the solid state.

Chapter 6 - Reduced Aib Content and Functional Groups A decapeptide with reduced Aib content and peptides incorporating Met or Phe were examined:

All peptides are helical in CDCl and MeOH. F 9 (with a single Aib residue) shows fragile helicity, collapsing in DMSO to extended conformations. Removing terminal protecting groups disrupts helicity completely.

Chapter 7 - Single Aib Residue Peptides Peptides containing a single Aib residue, even amidst many strand favoring residues, adopt helices in apolar solvents, though helicity collapses in DMSO. Aib significantly enhances solubility and helicity compared to analogous Aib free peptides.

Chapter 8 - Disulfide Linked vs. Acyclic Peptides Comparative studies on:

Boc Cys Val Aib Ala Leu Cys NHMe, and its cyclic disulfide counterpart

show:

cyclic peptide hairpin (Type II turn in solid state; Type I in solution), acyclic peptide helix in CDCl and hairpin in DMSO.

This highlights the role of disulfide crosslinks in stabilizing structures. Studies on acyclic cystine peptides further reveal antiparallel strand stabilization and segmental motions detected via NOEs.

Chapter 9 - “aa Corner” Peptide and Salt Bridge Design An 18 residue peptide modeling an aa corner motif was synthesized. Despite expectations, glycine adopts left handed helical torsion angles, favoring an overall helical rather than “aa corner” structure. Preliminary studies on a decapeptide designed to form interhelical salt bridges show promising trends.

Chapter 10 - Chemotactic Peptide Hybrid A nonapeptide incorporating the chemotactic tripeptide For Met Leu Phe at its N terminus exhibits biological activity comparable to the parent peptide. Structural analysis via CD and NMR suggests possible conformations for the bioactive form.

Chapter 11 - Summary The major findings of this investigation are summarized, highlighting:

the role of Aib in helicity, solubility, and conformational control, the structural effects of residue positioning, disulfide bonds, and protecting groups, the feasibility of assembling defined secondary structure modules for de novo protein design.