Evidence for the principle of minimal frustration in the evolution of protein folding landscapes.

Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates.

Intramolecular diffusion controls aggregation of the PAPf39 peptide.

The 39-residue fragment of human prostatic acidic phosphatase (PAP) is found in high concentrations in semen and easily form fibrils. Previous work has shown that fibrillization is accelerated with a deletion of the first 8, mostly charged residues and it was hypothesized that fibrillization depended on the dynamics of these peptides. To test this hypothesis we have measured the intramolecular diffusion of the full length and 8-residue deletion peptides at two different pHs and found a correlation with fibrillization lag time.

Modulation of folding energy landscape by charge-charge interactions: linking experiments with computational modeling.

The kinetics of folding-unfolding of a structurally diverse set of four proteins optimized for thermodynamic stability by rational redesign of surface charge-charge interactions is characterized experimentally. The folding rates are faster for designed variants compared with their wild-type proteins, whereas the unfolding rates are largely unaffected. A simple structure-based computational model, which incorporates the Debye-Hückel formalism for the electrostatics, was used and found to qualitatively recapitulate the experimental results.

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin.

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins.

Novel interactions of the TRTK12 peptide with S100 protein family members: specificity and thermodynamic characterization.

The S100 protein family consists of small, dimeric proteins that exert their biological functions in response to changing calcium concentrations. S100B is the best-studied member and has been shown to interact with more than 20 binding partners in a calcium-dependent manner. The TRTK12 peptide, derived from the consensus binding sequence for S100B, has previously been found to interact with S100A1 and has been proposed to be a general binding partner of the S100 family.

Equilibrium and kinetic studies of protein cooperativity using urea-induced folding/unfolding of a Ubq-UIM fusion protein.

Understanding the origins of cooperativity in proteins remains an important topic in protein folding. This study describes experimental folding/unfolding equilibrium and kinetic studies of the engineered protein Ubq-UIM, consisting of ubiquitin (Ubq) fused to the sequence of the ubiquitin interacting motif (UIM) via a short linker. Urea-induced folding/unfolding profiles of Ubq-UIM were monitored by far-UV circular dichroism and fluorescence spectroscopies and compared to those of the isolated Ubq domain.

Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences.

How the primary sequence of a protein encodes conformational preferences that operate early in folding to promote efficient formation of the correct native topology is still poorly understood. To address this issue, we have prepared a set of yeast iso-1-cytochrome c variants that contain polyalanine inserts ranging from 6 to 30 residues in length near the N terminus of the protein. We study the thermodynamics and kinetics of His-heme loop formation in the denatured state at 3 and 6 M guanidine-HCl concentration.

Thermodynamics of loop formation in the denatured state of rhodopseudomonas palustris cytochrome c': scaling exponents and the reconciliation problem.

The observation that denatured proteins yield scaling exponents, nu, consistent with random-coil behavior and yet can also have pockets of residual or nonrandom structure has been termed the "reconciliation problem". To provide greater insight into the denatured state of a foldable sequence, we have measured histidine-heme loop formation equilibria in the denatured state of a class II c-type cytochrome, cytochrome c' from Rhodopseudomonas palustris. We have prepared a series of variants that provide His-heme loop stabilities, pK(loop)(His), for loop sizes ranging from 10 to 111 residues at intervals of 7 to 11 residues along the sequence of the protein.

Importance of contact persistence in denatured state loop formation: kinetic insights into sequence effects on nucleation early in folding.

Protein folding is dependent on the formation and persistence of simple loops early in folding. Ease of loop formation and persistence is believed to be dependent on the steric interactions of the residues involved in loop formation. We have previously investigated this factor in the denatured state of iso-1-cytochrome c using a five-amino-acid insert in front of a unique histidine in the N-terminal region of the protein.

Competition between reversible aggregation and loop formation in denatured iso-1-cytochrome c.

The competition between intramolecular histidine-heme loop formation and ligand-mediated oligomer formation in the denatured state is investigated for two yeast iso-1-cytochrome c variants, AcH26I52 and AcA25H26I52. Besides the native His 18 heme ligand, both variants contain a single His at position 26. The AcA25H26I52 variant has Pro 25 mutated to Ala.

Sequence composition effects on denatured state loop formation in iso-1-cytochrome c variants: polyalanine versus polyglycine inserts.

Protein folding is dependent on the formation and persistence of simple loops during the earliest events of the folding process. Ease of loop formation and persistence is believed to be dependent on the steric properties of the residues involved in loop formation. We have investigated this conformational factor in the denatured state of iso-1-cytchrome c using a five alanine insert in front of a unique histidine in the N-terminal region of the protein.