Robert L. Baldwin

How Hofmeister ion interactions affect protein stability.

Model compound studies in the literature show how Hofmeister ion interactions affect protein stability. Although model compound results are typically obtained as salting-out constants, they can be used to find out how the interactions affect protein stability. The null point in the Hofmeister series, which divides protein denaturants from stabilizers, arises from opposite interactions with different classes of groups: Hofmeister ions salt out nonpolar groups and salt in the peptide group. Theories of how Hofmeister ion interactions work need to begin by explaining the mechanisms of these two classes of interactions. Salting-out nonpolar groups has been explained by the cavity model, but its use is controversial. When applied to model compound data, the cavity model 1) uses surface tension increments to predict the observed values of the salting-out constants, within a factor of 3, and 2) predicts that the salting-out constant should increase with the number of carbon atoms in the aliphatic side chain of an amino acid, as observed. The mechanism of interaction between Hofmeister ions and the peptide group is not well understood, and it is controversial whether this interaction is ion-specific, or whether it is nonspecific and the apparent specificity resides in interactions with nearby nonpolar groups. A nonspecific salting-in interaction is known to occur between simple ions and dipolar molecules; it depends on ionic strength, not on position in the Hofmeister series. A theory by Kirkwood predicts the strength of this interaction and indicates that it depends on the first power of the ionic strength. Ions interact with proteins in various ways besides the Hofmeister ion interactions discussed here, especially by charge interactions. Much of what is known about these interactions comes from studies by Serge Timasheff and his co-workers. A general model, suitable for analyzing diverse ion-protein interactions, is provided by the two-domain model of Record and co-workers.

Is protein folding hierarchic? I. Local structure and peptide folding

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Nevertheless, we argue that both classes fold hierarchically and that folding begins locally. If this is the case, then the secondary structure of a protein is determined largely by local sequence information. Experimental data and theoretical considerations support this argument. Part I of this article reviews the relationship between secondary structures in proteins and their counterparts in peptides.

Cation-induced toroidal condensation of DNA: Studies with Co3+(NH3)6

Abstract The polyamines spermidine (3+) and spermine (4+) are known to cause a co-operative intramolecular condensation of T7 or λ phage DNAs in which the DNA assumes a compact toroidal conformation (Gosule & Schellman, 1976,1978). We show here that an inert trivalent metal ion complex, Co 3+ (NH 3 ) 6 , also causes DNA condensation and that DNA condensation in aqueous solution is caused by cations of charge 3+ or more. The DNA products of polyamine-induced and of cobalt hexamine-induced condensation have similar toroidal conformations as judged by electron microscopy and both have the circular dichroism spectrum of DNA B-form, in contrast to the ψ DNA condensates studied by Maniatis et al. (1974). Monovalent and divalent cations (Na + , Mg 2+ ) reverse DNA condensation. Competition between inducing (3+ or 4+) and reversing (1+ or 2+) cations follows the ion-exchange behavior outlined in Mannings (1978) theory of atmospheric cation binding to DNA. Our results are consistent with the suggestion of Manning (1978) and of Wilson & Bloomfield (1979) that DNA condensation can occur when a critical fraction of the DNA phosphate charge has been neutralized by cations adsorbed to the DNA. We suggest further that cation-induced DNA condensation in aqueous solution results from cation crosslinking: electrostatic bridging of adjacent helices by trivalent or higher valence cations. Transition curves for DNA condensation have been measured by the increase in light-scattering, using a photon-counting fluorimeter. To ensure that equilibrium is reached, condensation has been studied in both the forward and reverse directions, by using either Na + or Mg 2+ to reverse the reaction. The kinetics of condensation are slow in the forward direction, in the time range of minutes to hours, and become much slower as the DNA concentration is increased. Reversal of condensation by Na + or Mg 2+ occurs more rapidly, in seconds to minutes, and the transition midpoints are essentially independent of DNA concentration. At DNA concentrations below 1 μ m -phosphate, the kinetics of condensation and of de-condensation are comparable in rate. We suggest that intermolecular DNA contacts compete with, and slow down, intramolecular condensation. Equilibrium data for transition midpoints are obtained in either the forward or reverse direction at sufficiently low DNA concentrations; at higher DNA concentrations, equilibrium is reached in the reverse but not in the forward direction. Phase diagrams for condensation (plots of log [Co 3+ (NH 3 ) 6 ] versus log [Na + ] or log [Mg 2+ ] at the transition midpoint) have been obtained from studies of de-condensation by Na + or Mg 2+ . These plots have a slope of +1 when either Co 3+ (NH 3 ) 6 , spermidine (3+) or spermine (4+) is used to induce condensation. As shown by Wilson & Bloomfield (1979), a slope of +1 is consistent with DNA condensation occurring when a critical fraction of DNA charge has been neutralized, as calculated by Mannings (1978) theory. Two additional results are presented, which bear on the problem of toroidal DNA condensation. (1) Condensation occurs more readily at high temperatures. (2) Restriction fragments as short as 400 base-pairs form toroids by intermolecular condensation, which are similar in diameter and appearance to the intramolecular condensates formed by λ DNA.

Polyproline II structure in a sequence of seven alanine residues

A sequence of seven alanine residues—too short to form an α-helix and whose side chains do not interact with each other—is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The 3JHNα coupling constants of individual alanine residues have been measured from 2 to 56°C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2°C and the content of β strand is increased by approximately 10% at 55°C relative to that at 2°C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer “random coil” peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.

Energetics of DNA twisting: I. Relation between twist and cyclization probability*

The twisting potential of DNA has been determined directly by a method that measures the cyclization probability or j-factor of EcoRI restriction fragments as a function of DNA twist. The cyclization probability is proportional to Kc, the equilibrium constant for cyclization of the restriction fragment via its cohesive ends (Shore et al., 1981). Here we vary the twist of the DNA by making small internal additions to or deletions from a 242 bp EcoRI restriction fragment. A series of 12 DNA molecules has been studied, which range in length from 237 to 254 bp. The cyclization probability is measured from the rates of covalent closure by phage T4 DNA ligase of two systems: (1) a linear restriction fragment in equilibrium with its cyclized form and (2) half molecules (cut by a blunt-end endonuclease) in equilibrium with joined half molecules. The striking result is that, in this DNA size range, the j-factor depends strongly on the fractional twist: the difference between the total helical twist and the nearest integer. Thus j depends in an oscillatory manner on DNA length between 237 and 254 bp with a period of about 10 bp. These data give the free energy of DNA twisting as a function of twist. The curve of j versus DNA length can be fitted to a harmonic twisting potential with a torsional constant of C = 2.4 X 10(-19) erg cm. This value is in reasonable agreement with different estimates of C made by Barkley & Zimm (1979: C = 1.8 X 10(-19) to 4.1 X 10(-19) erg cm) and is somewhat larger than the value obtained resulting from the kinetics of DNA twisting measured by fluorescence depolarization of ethidium intercalated into DNA (C = 1.4 X 10(-19) erg cm; Millar et al., 1982; Thomas et al., 1980) or from spin label studies (Hurley et al., 1982). Our experiments provide a direct measurement of the torsional free energy and they show that the DNA twisting potential is symmetric. Our experiments also indicate that the DNA helix is continuous, or nearly so, in a nicked circle; presumably this happens because the DNA stacking interaction maintains the double helix in register across a single-strand nick. As a consequence, the twist of a singly nicked DNA circle is integral for small (approximately equal to 250 bp) planar DNA circles and there is a change in twist upon cyclization.(ABSTRACT TRUNCATED AT 400 WORDS)

Is protein folding hierarchic? II. Folding intermediates and transition states

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Evidence from folding intermediates and transition states suggests that folding begins locally, and that the formation of native secondary structure precedes the formation of tertiary interactions, not the reverse. Some notable examples in the literature have been interpreted to the contrary. For these examples, we have simulated the local structures that form when folding begins by using the LINUS program with nonlocal interactions turned off. Our results support a hierarchic model of protein folding.

STABILITY OF ALPHA -HELICES

Publisher Summary This chapter focuses on the mechanism of helix formation in an isolated peptide and the factors that determine the stability of a peptide helix. Helix propensities are considered together with N-cap and C-cap propensities, because measurement of helix propensities requires knowing values of the N-cap and C-cap propensities, and vice versa. The chapter considers side-chain interactions: these include both the interaction of a charged side chain with the helix macrodipole and specific interactions between a particular pair of side chains, such as ion pair and H-bond interactions. Measurement of these interactions is of interest for two reasons: their values are needed to relate the stability of a peptide helix to its amino acid composition and sequence; and peptide helices provide one of the best systems, and probably the most sensitive system, for quantifying the energetics of side-chain interactions. It also considers briefly the present status of the Chou-Fasman hypothesis and the relation between the mechanism of α-helix formation in peptides and proteins. It is necessary to use helix-coil transition theory to understand the populated intermediates and to analyze the energetics of helix formation. The two closely related theories of α-helix formation are the Zimm-Bragg theory and the Lifson-Roig theory.

The nature of protein folding pathways: The classical versus the new view

SummaryPulsed hydrogen exchange and other studies of the kinetic refolding pathways of several small proteins have established that folding intermediates with native-like secondary structures are well populated, but these studies have also shown that the folding kinetics are not well synchronized. Older studies of the kinetics of formation of the native protein, monitored by optical probes, indicate that the folding kinetics should be synchronized. The model commonly used in these studies is the simple sequential model, which postulates a unique folding pathway with defined and sequential intermediates. Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates accumulate because of kinetic traps caused by partial misfolding. Recent experiments with cytochrome c lend support to this ‘new view’ of folding pathways. These different views of the folding process are discussed. Misfolding and consequent slowing down of the folding process as a result of cis-trans isomerization about prolyl peptide bonds in the unfolded protein are well known; isomerization occurs before refolding is initiated. The occurrence of equilibrium intermediates on the kinetic folding pathways of some proteins, such as α-lactalbumin and apomyoglobin, argues that these intermediates are not caused by kinetic traps but rather are stable intermediates under certain conditions, and this conclusion is consistent with a sequential model of folding. Folding reactions with successive kinetic intermediates, in which late intermediates are more highly folded than early intermediates, indicate that folding is hierarchical. New experiments that test the predictions of the classical and the new views are needed.

Analysis of casein fractions by zone electrophoresis in concentrated urea

Abstract Disaggregation of casein by concentrated solutions of urea reveals many new components upon starch-gel electrophoresis. When individual bands are cut out of the gel, eluted, and the protein again analyzed by electrophoresis, the bands appear in the same positions as before: therefore single components do not produce multiple bands. Samples of whole casein prepared in three different ways (acid precipitation, Na 2 SO 4 precipitation, sedimentation of the casein micelles) give the same starch-gel analyses; thus it is not likely that new components are produced during preparation. Caseins from three individual cows (two of β-lactoglobulin type A, one of type B) show the same starch-gel patterns as casein from pooled milk. A component can be identified by the relative distance its band has moved from the starting slot, and so the components are numbered by their relative band positions. Most of the new components are found in the α fraction of casein; the β fraction is relatively homogeneous. The system of alcohol fractionation used by Hipp et al. to prepare α-, β-, and γ -caseins achieves some separation of the components within the α fraction; samples of “ α -casein” prepared by different methods are likely to differ. Study of the “soluble casein” fractions of Waugh and von Hippel shows an enrichment of κ -casein in fraction S, but not a clear separation. Starch-gel analysis has been used to study the action of rennin on whole casein. The results confirm the conclusion of Waugh and von Hippel that κ -casein is the only component attacked in the primary reaction.

HELIX--RANDOM COIL TRANSITIONS IN DNA HOMOPOLYMER PAIRS.

Conditions have been found where simple helix-random coil transitions occur for each of the DNA homopolymer pairs dGdC, † dIdC and d I d B C ¯ and it is shown that these correspond to the melting of dG : dC, dI : dC and dI : d B C ¯ base pairs. The complementary strands of the homopolymer pairs can be separated and identified by density-gradient centrifugation in Cs2SO4 at alkaline pH. Although this technique demonstrates complete separation for homopolymer pairs, incomplete separation results when dAT : d A B U ¯ copolymer hybrids are studied in the same way. The isolated homopolymer strands have been used to re-form dIdC and d I d B C ¯ ; mixing experiments show maximum interaction for a purine: pyrimidine mole ratio of 1, as expected for dI : dC and dI : d B C ¯ base pairs. The changes in absorbancy and in viscosity which accompany the thermal transition are qualitatively like those of natural DNAs. However, there are some interesting quantitative differences. The width of the melting zone is independent of salt concentration over at least a 20-fold range. This contrasts with the behavior of natural DNAs whose melting curves broaden in low salt concentrations, and also contrasts with the dAT copolymer which shows a sharp transition in low-salt but a broad transition in high salt concentrations. The thermal stability (Tm) of each homopolymer pair has been determined over a wide range of ionic strength. After melting, the homopolymer pairs re-form on cooling below Tm. The kinetics are second-order for most of the reaction and the initial rate increases with Tm–T but decreases as the salt concentration is reduced, much like the results of Ross & Sturtevant (1960) for rA : rU. In contrast, re-formation of dAT : dAT is rapid even at low ionic strength, presumably because the double helix re-forms from a single strand. The alkaline transition for each homopolymer pair has also been investigated. At constant ionic strength and temperature, pHm is determined by the base pair stability and by the pKa′ of the base titrated during the transition; thus there is a relation between pHm–pKa′ and Tm. On cooling through the transition zone, the viscosity rises as the homopolymer pair re-forms. However, the viscosity continues to increase gradually on further cooling down to 0°C; this probably is due to end-to-end aggregation of helical units.