A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains

Pochapsky, Thomas C.; Gopen, Quinton

doi:10.1002/pro.5560010610

Cited by 23 publications

(23 citation statements)

References 39 publications

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“…The error estimate of k40 cal/mol associated with AAG" bind was obtained by a uniform application of the 5% relative standard deviation to all of the adsorption isotherm data. The magnitude of this error is comparable to the resolution limit of 30 caYmol reported by Pochapsky and Gopen (1992) in their chromatographic binding studies.…”

Section: Binding Isotherms For Site Identificationsupporting

confidence: 75%

“…Our development of the SIC technique was motivated by the chromatographic studies of Pochapsky and Gopen (1992) and Chiancone and co-workers (1992). Pochapsky and Gopen studied the retention of amino acid derivatives on a stationary phase covalently modified with a functional group mimicking the side chain of phenylalanine.…”

Section: Self-interaction Chromatographymentioning

confidence: 99%

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Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Patro

Przybycien

2000

Biotechnol. Bioeng.

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We describe a new protein characterization technique called self‐interaction chromatography (SIC), which exploits the specificity of protein–protein interactions that is common to protein aggregates and enables the rapid screening of protein formulation additives as physical stabilizers against aggregation. This technique also enables the identification of specific interaction sites and the determination of their relative importance for self‐association. Mannitol, glycine, and dextran 40 were tested for their stabilizing effect toward the model protein lysozyme. Dextran 40 exhibited a poor stabilizing effect. While mannitol stabilized both the native and acid‐denatured forms of lysozyme, glycine stabilized the native form with respect to the denatured species. These results are in good agreement with findings in the formulation literature. The SIC shows tremendous potential as a rapid formulation development tool. We also screened two putative interaction sites for involvement in the self‐association of lysozyme and estimated the associated binding energies using a binding isotherm model that we developed. The sites screened consisted of residues 41–48 and 125–128 and were selected based on their apparent importance in forming crystal contacts in several different crystal forms of lysozyme. Of the two sites, only residues 125–128 were found to influence self‐association under the conditions we employed. Because the success of this technique depends on the exploitation of self‐interactions between native species, several important applications are also suggested such as separating native from misfolded or variant species and probing site utilization in aggregation versus crystallization phenomena. © 1996 John Wiley & Sons, Inc.

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Section: Binding Isotherms For Site Identificationsupporting

confidence: 75%

Section: Self-interaction Chromatographymentioning

confidence: 99%

Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Patro

Przybycien

2000

Biotechnol. Bioeng.

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“…This distinction is likely to reflect experimentally observed qualitative differences in the thermodynamics of interaction between aromatic and aliphatic sidechains. [30][31][32][33] For hydrophilic residues, which appear to the left of the reference curve, charged amino acids (K, R, E, and D) also have a s > 1.35 and can therefore be distinguished from uncharged residues (Q, P, N). Note, from the values shown in Table I or the isohysrophobicity lines in Figure 10(b), that a single hydrophobicity parameter, h s or a s h s * , is unable to distinguish not only aromatic from aliphatic residues (e.g., h F > h I > h W > h L > h Y ), but also hydrophilic charged from uncharged (e.g., h K < h Q < h R ).…”

Section: Discussionmentioning

confidence: 99%

Description of atomic burials in compact globular proteins by Fermi‐Dirac probability distributions

et al. 2006

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We perform a statistical analysis of atomic distributions as a function of the distance R from the molecular geometrical center in a nonredundant set of compact globular proteins. The number of atoms increases quadratically for small R, indicating a constant average density inside the core, reaches a maximum at a size-dependent distance R(max), and falls rapidly for larger R. The empirical curves turn out to be consistent with the volume increase of spherical concentric solid shells and a Fermi-Dirac distribution in which the distance R plays the role of an effective atomic energy epsilon(R) = R. The effective chemical potential mu governing the distribution increases with the number of residues, reflecting the size of the protein globule, while the temperature parameter beta decreases. Interestingly, betamu is not as strongly dependent on protein size and appears to be tuned to maintain approximately half of the atoms in the high density interior and the other half in the exterior region of rapidly decreasing density. A normalized size-independent distribution was obtained for the atomic probability as a function of the reduced distance, r = R/R(g), where R(g) is the radius of gyration. The global normalized Fermi distribution, F(r), can be reasonably decomposed in Fermi-like subdistributions for different atomic types tau, F(tau)(r), with Sigma(tau)F(tau)(r) = F(r), which depend on two additional parameters mu(tau) and h(tau). The chemical potential mu(tau) affects a scaling prefactor and depends on the overall frequency of the corresponding atomic type, while the maximum position of the subdistribution is determined by h(tau), which appears in a type-dependent atomic effective energy, epsilon(tau)(r) = h(tau)r, and is strongly correlated to available hydrophobicity scales. Better adjustments are obtained when the effective energy is not assumed to be necessarily linear, or epsilon(tau)*(r) = h(tau)*r(alpha,), in which case a correlation with hydrophobicity scales is found for the product alpha(tau)h(tau)*. These results indicate that compact globular proteins are consistent with a thermodynamic system governed by hydrophobic-like energy functions, with reduced distances from the geometrical center, reflecting atomic burials, and provide a conceptual framework for the eventual prediction from sequence of a few parameters from which whole atomic probability distributions and potentials of mean force can be reconstructed.

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“…The hydrophobic energy function, upon which this study is based, is no more than a lattice implementation of the physical assumption that conformational free energy of real proteins can be computed from solvent accessible surface areas of different types, such as aliphatic, aromatic and polar, combined with unit free energies of hydration, which can in principle be obtained from partition experiments (24)(25)(26)(27). Recent estimates for free energy changes involved in specific pairwise interactions obtained from HPLC experimental data are not inconsistent with this simple scheme (28,29). A recent decomposition (30) of the Miyazawa and Jerningan potential (31) has also suggested that hydrophobicity can largely explain the statistical distribution of amino acid contacts in protein structures.…”

Section: Discussionmentioning

confidence: 99%

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

Araújo

1999

Proc. Natl. Acad. Sci. U.S.A.

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The present study explores a "hydrophobic" energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its "hydrophobicity" and the number of contacts it makes, i.e., E(h,c) = -Sigma N/i=1 c(i)h(i) = -(h.c) is the negative scalar product between two vectors in N-dimensional cartesian space: h = (h1,., hN), which represents monomer hydrophobicities and is sequence-dependent; and c = (c(1),., c(N)), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c, the best sequence is a vector h on the direction upon which the projection of c - c is maximal, where c is the diagonal vector with components equal to c, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

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A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains

Cited by 23 publications

References 39 publications

Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Description of atomic burials in compact globular proteins by Fermi‐Dirac probability distributions

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

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