Increasing Protein Conformational Stability by Optimizing β-Turn Sequence

Glycine and proline residues are frequently found in turn and loop structures of proteins and are believed to play an important role during chain compaction early in folding. We investigated their effect on the dynamics of intrachain loop formation in various unstructured polypeptide chains. Loop formation is significantly slower around trans prolyl peptide bonds and faster around glycine residues compared to any other amino acid. However, short loops are formed fastest around cis prolyl bonds with a time constant of 6 ns for end-to-end contact formation in a four-residue loop. Formation of short loops encounters activation energies in the range of 15 to 30 kJ/mol. The altered dynamics around glycine and trans prolyl bonds can be mainly ascribed to their effects on the activation energy. The fast dynamics around cis prolyl bonds, in contrast, originate in a higher Arrhenius pre-exponential factor, which compensates for an increased activation energy for loop formation compared to trans isomers. All-atom simulations of proline-containing peptides indicate that the conformational space for cis prolyl isomers is largely restricted compared to trans isomers. This leads to decreased average end-to-end distances and to a smaller loss in conformational entropy upon loop formation in cis isomers. The results further show that glycine and proline residues only influence formation of short loops containing between 2 and 10 residues, which is the typical loop size in native proteins. Formation of larger loops is not affected by the presence of a single glycine or proline residue.

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.

Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry.

The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes was investigated employing alpha- and gamma-chymotrypsin. As a measure of the conformational stability the melting temperature T(m) was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier-transform infrared spectroscopy employing two regions sensitive to protein structure, the amide-I (1600-1700 cm(-1)) and amide-III (1215-1335 cm(-1)). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the alpha-helix and a significant increase in the beta-sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization-induced structural changes by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization-induced structural alterations, we conducted co-lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air-drying experiment, mainly causes protein structural alterations.

The side chain of proline is made of an aliphatic five-membered pyrrolidine ring. The side chain is unique among protein-constituting amino acids in that its terminal Cδ is covalently bonded to the preceding pep tide bond nitrogen. The polypeptide backbone at this point has no amide hydrogen for use as a donor in hydrogen bonding. The pyrrolidine ring makes rigid constraints on the rotation about the NCα bond of the peptide backbone and drastically fixes the angle of rotation to approximately −58° (1). The conformational energy of a proline residue depends largely on the angle ψ of rotation about the CαC bond of the peptide backbone. The proline residue, when isolated from other proline residues in a polypeptide chain, has two energy minima at ψ=−55° and ψ=l45° (1). The bulky pyrrolidine ring restricts the available conformational space of the preceding residue in the polypeptide chain if its side chain extends at least to Cβ (1, 2). The peptide bond (Xaai−1Proi) preceding a proline residue, unlike those preceding other amino acid residues, might not be expected to have double bond character due to the lack of an imide hydrogen (3). The Xaai-1Proi peptide bond is more likely to adopt the cis rather than the trans configuration, compared with other peptide bonds (probability: 0.10.3 vs. <10-3) (2, 3). The difference in the standard free energy ∆G° between cis and trans Xaai−1Proi peptide bonds is on the order of +8 to −8 kJ/mol (1 J=0.239 cal) (4), and the activation energy for the cis-trans isomerization is about 54 kJ/mol, which is far less than 84 kJ/ mol for other peptide bonds (2, 3). However, the rate of the cis-trans isomerization is very slow for the Xaai−1 Proi peptide bond, with a half-time of 20 min at 0°C, compared with 1012 s−1 for other peptide bonds (3). The above-mentioned exceptional properties of proline impose significant constraints on the polypeptide conformation of proteins, their folding processes, and their functions (2-9). Thus, proline tends to be a conserved residue (2, 10). In 1988, the frequency of proline was determined to be 5.1 mol% among 1021 unrelated proteins of known sequence (11). This frequency is comparable with that (4.8 mol% among 393 unrelated protein sequences) reported by Chakrabarti and Pal (9). In 1982, we found a big difference in the proline content between oligo-1,6-glucosidases from Bacillus cereus ATCC7064 and B. thermoglucosidasius KP1006 (12). At that time, there was no report about the relationship between proline frequency and thermostability, except one made by Crabb etal. (13) on D-glyceraldehyde-3-phosphate dehydrogenases from the facultative thermophile B. coagulans and the obligate thermophile B. stearothermophilus. On sequence comparison, they proposed a proline residue in a solvent-exposed variable loop of the B. stearothermophilus enzyme to contribute to its higher thermal stability. In 1987, we found a strong correlation between the number of proline residues and thermostability (expressed as tm*) for five Bacillus oligo-1,6-glucosidases (2, 14-17). Based on this finding, we proposed a general principle for increasing protein thermostability, known as the Proline Rule (16, 18). The rule states that globular proteins can be thermostabilized by increasing the frequency of proline in the second position (i+1) of β-turns.† A direct proof for the proposal was given by Matthews et al. (19). These authors increased the thermostability of bacteriophage T4 lysozyme by replacing an alanine residue with a proline residue in one of the β-turns to decrease the backbone entropy of the unfolded molecule. From 1994 to 1998, we cumulatively thermostabilized B. cereus oligo-1,4glucosidase through stepwise addition of proline residues in 12 separate positions, namely, four i+1 positions in β-turns, four N1 positions in the first turns of α-helices, and four positions in coils (21). The validity of the Proline Rule has been confirmed by sitedirected mutagenesis on various proteins (21-44). Many findings correlating proline residues to thermostability have been reported in numerous proteins from microorganisms including hyperthermophiles (45-63). Directed evolution for protein thermal stabilization has been found to include proline substitutions (64-67). It is furthermore interesting that many psychrophilic proteins have a low number of proline residues (68-72). In 1999, Suzuki (38) proposed the Proline Rule in refined form. The Proline Rule suggests an evolutionary rule for protein thermal adaptation (38, 73-75) and a paradigm for engineering protein thermostability (19, 21-23, 38). This paper describes three steps that we have taken to examine and refine this rule (38).

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

The molecular thermodynamic model studied is based on the two‐state mechanism of inactivation, in which only native folded and polymorphous unfolded protein forms are present at equilibrium. The influence of solvent on protein stability is described in terms of perturbation of the protein distribution between the two conformational states. An expression derived for the chemical potential of the protein accounts for conformational changes, ideal mixing effects, and interaction of the protein with the surrounding medium.Thermal unfolding of lysozyme was then studied in the absence or presence of hydroxylic compounds. Ultraviolet difference spectroscopy was used to monitor the conformational changes induced by heating and to determine the melting temperature of the protein. The additives investigated are ethanol, glycols, and natural osmolytes. Media containing ethanol and glycols destabilized lysozyme, whereas sugars increased the conformational stability of the protein. For all of the systems examined the melting temperature was linearly related to the surface tension of the mixed solvent, supporting the ability of the model to describe the influence of the solvent and composition on lysozyme unfolding. Model predictions agreed fairly well with published differential scanning calorimetric data. The influence of hydroxylic additives on protein's conformational stability does not extend to any special property of these components, but to their ability to perturb the surface tension of water. This model can be used to interpret and correlate thermal unfolding data and to solve the problem of protein stabilization.

Apparently contradictory statements about the thermodynamics of aqueous protein solutions and of hydrophobic effect are quoted and discussed. Some credibility is found in the divergent points of view and it is pointed out that they focus attention on different aspects of the complicated conditions in aqueous solutions, some of which are more important than others for the stability of protein conformations.

Proteins must fold to a globular conformation to carry out the most important tasks in living organisms. The folded, biologically active conformation of a protein is only marginally more stable than the unfolded, inactive conformations. Thus, making proteins more stable is important in medicine and basic research. The major destabilising force that must be overcome is conformational entropy. The major stabilising forces are the hydrophobic effect and hydrogen bonding. The ionisable side chains of amino acid residues may also contribute favourably to protein stability through attractive charge–charge interactions, ion pair formation or the formation of hydrogen bonds when such groups are buried in the protein interior. Replacing a nonproline or nonglycine residue in a β‐turn can also significantly increase protein stability. Key Concepts: Under physiological conditions, a folded protein is ∼20 to 60 kJ mol –1 more stable than unfolded forms. The major destabilising force to protein folding is conformational entropy, which contributes ∼7 kJ mol −1 per residue. The major stabilising forces are the hydrophobic effect, where the burying of each –CH 2 – contributes ∼−5 kJ mol −1 , and hydrogen bonding, especially buried intramolecular hydrogen bonds, which may contribute ∼−7 kJ mol −1 per bond. The ionisable side chains of amino acid residues may contribute favourably to protein stability through attractive charge–charge interactions, ion pair formation and hydrogen bonding when the ionisable group is buried in the protein interior. Replacing a nonproline or nonglycine residue in a β‐turn may increase protein stability.

Interpolyelectrolyte complexes of lysozyme with short poly[di(carboxylatophenoxy)phosphazene]. Binding energetics and protein conformational stability

Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.

The left-handed polyproline II helix (PPII) is believed to be the preferred conformation for proline-rich regions of sequence in proteins. Such regions have been postulated to be protein-protein interaction domains. The formation of this structure is studied here using simple Monte Carlo computer simulations employing the hard sphere potential. It is found that polyproline sequences adopt only the PPII structure in the simulations. Non-proline, non-glycine residues inserted as guests into polyproline host peptides are conformationally restricted by the following proline residues and tend to be part of the PPII helix. It is found through insertion of two alanine residues into polyproline that the PPII structure is not propagated through more than one non-proline residue. This finding calls into question the hypothesis that proline-rich regions will preferentially adopt this structure since many such sequences are comprised of less than 50% proline residues. Proteins 33:218–226, 1998. © 1998 Wiley-Liss, Inc.

Conformational stability of proteins and peptide-peptide interactions in the presence of carbohydrates

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

To explore the effect of an external electrostatic field (EEF) on the stability of protein conformations, the molecular dynamic modeling approach was applied to evaluate the effect of an EEF along the x or y direction on a water cluster containing a parallel or antiparallel β sheet structure. The β sheet structure contained two strands with a (Gly)3 sequence separated by a distance d along the x direction. The mean forces between the two strands along the x direction were computed from the trajectories of molecular dynamics simulations. In the absence of the EEF, the forces between the two strands in vacuum were repulsive and attractive in the parallel and antiparallel β sheet structures, respectively. In contrast, the mean forces between the two strands in water were attractive in both the parallel and antiparallel β sheet structures. This is because the electric interactions between the two strands were shielded by water, and the hydrophobic effect dominated the interaction between the two strands. When an EEF >50 MV/cm was applied to the water cluster, the attractive force between the two strands in the parallel and antiparallel β sheet structures decreased and increased, respectively. Further, the binding affinity between the two strands in the parallel and antiparallel β sheet structures also decreased and increased, respectively. This is because the large EEF leads to dielectric saturation, and consequently reduces the effects of the dielectric shielding and hydrophobic interactions.