Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is approximately 1 x 10(8) M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron-porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme-protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme-globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is approximately 1 x 10(14) M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 10(5)-10(7). Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 10(3)-10(4). Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 10(3)-10(4).

The translational roles of the Shine-Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied. The Shine-Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence. Each Shine-Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long. Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG. Translation was also affected by residues 3' to the initiation codon. The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon. The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation.

The proximal bond between the iron atom of the heme group and the N epsilon of histidine F8 in myoglobin (Mb) and hemoglobin (Hb) is presumed to be an important determinant of heme binding, protein structure, and oxygen binding. Here a system is described in which the proximal ligand is provided intermolecularly by the histidine side chain mimic imidazole. The proximal ligand of sperm whale Mb is replaced with glycine (H93G) using site-directed mutagenesis. The addition of imidazole to Escherichia coli expressing this gene reconstitutes myoglobin function. H93G Mb purified in the presence of imidazole is spectroscopically similar to wild-type Mb in combination with a wide variety of distal ligands. The crystal structure of H93G Mb, determined in the presence of imidazole, reveals that an imidazole molecule is bonded to the heme iron on the proximal side, substituting in trans for the side-chain function of the proximal histidine of wild-type Mb. Although H93G Mb is similar in spectroscopic and gross structural detail to wild-type Mb, subtle differences exist in the orientation of imidazole with respect to the heme group. trans-Complementation of proximal ligand function will allow the proximal bond in hemoproteins to be chemically substituted beyond the limits of the genetic code.

Energy landscapes have been used to conceptually describe and model protein folding but have been difficult to measure experimentally, in large part because of the myriad of partly folded protein conformations that cannot be isolated and thermodynamically characterized. Here we experimentally determine a detailed energy landscape for protein folding. We generated a series of overlapping constructs containing subsets of the seven ankyrin repeats of the Drosophila Notch receptor, a protein domain whose linear arrangement of modular structural units can be fragmented without disrupting structure. To a good approximation, stabilities of each construct can be described as a sum of energy terms associated with each repeat. The magnitude of each energy term indicates that each repeat is intrinsically unstable but is strongly stabilized by interactions with its nearest neighbors. These linear energy terms define an equilibrium free energy landscape, which shows an early free energy barrier and suggests preferred low-energy routes for folding.

We give a quantitative description of the urea- and acid-induced transitions of apomyoglobin at 0 degree C and 2 mM sodium citrate. Our data consist of two series of unfolding curves: (1) acid-induced unfolding carried out in the presence of various concentrations of urea and (2) urea-induced unfolding at various pH values. A three-state equation is derived which relates the stability of three different conformations of apomyoglobin (native [N], unfolded [U], and intermediate [I]) as a function of urea and of pH. This equation fits our data reasonably well. The parameters which give the best fit have both thermodynamic and structural implications for N, I, and U. Specifically, I is closer in Gibbs energy to U than to N, indicating that side-chain packing results in much of the stability of native protein structure. The equilibria between N and I and between I and U are equally sensitive to urea, suggesting that much of the surface of I is inaccessible to solvent. The acid-induced transition in which N unfolds can be described as the result of titration of approximately two histidines with low pKaS in N. Under physiological conditions (neutral pH, no urea) I is the most stable non-native conformation.

To provide a model system for understanding how the unfolding of protein a-helices by urea contributes to protein denaturation, urea unfolding was measured for a homologous series of helical peptides with the repeating sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying from 14 to 50 residues. The dependence of the helix propagation parameter of the Zimm-Bragg model for helix-coil transition theory (s) on urea molarity ([urea]) was determined at 0°C with data for the entire set of peptides, and a linear dependence ofIn s on [urea] was found. The results were fitted by the binding-site model and by the solvent-exchange model for the interaction of urea with the peptides. Each of these thermodynamic models is able to describe the data quite well and we are not able to discern any difference between the ability of each model to fit the data. Thus a linear relation, In s = In so -(m/R7). [urea], fits the data for av-helix unfolding, just as others have found for protein unfolding. When the m value determined here for a-helix unfolding is multiplied by the number of helical residues in partly helical protein molecules, the resulting values agree within a factor of 2 with observed m values for these proteins. This result indicates that the interaction between urea and peptide groups accounts for a major part of the denaturing action of urea on proteins, as predicted earlier by some model studies with small molecules.We use helix-coil theory to examine how the a-helix to random coil transition depends on urea molarity for a homologous series of peptides. We address four specific questions. (i) Does urea denature proteins by interacting with the peptide group (1-3) or by solubilizing nonpolar side chains, especially aromatic groups (4-6)? (ii) Can the urea dependence of the a-helix unfolding reaction be explained quantitatively by the popular models [binding-site model (7, 8) and solventexchange model (9, 10)] for the solvent denaturation of proteins? (iii) Can the urea-induced unfolding transition of the a-helix be used as a simple model system to discriminate between these thermodynamic models? (iv) Can the dependence on urea molarity of native protein and molten globule unfolding be considered to result from the action of urea on protein a-helices and other elements of secondary structure?The observation that short, alanine-based peptides form monomeric helices in aqueous solution (11) has permitted the direct determination of the energetics of helix formation [see review by Scholtz and Baldwin (12)]. Thermally induced helix-coil transitions in these peptides have been shown by spectroscopy and calorimetry (13,14) to conform to helix-coil theory, which predicts that peptide helix unfolding is cooperative and multistate (15,16), and have provided estimates of Gibbs energies and enthalpies of helix unfolding (13, 14).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely t...

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that ⌬G°for unfolding depends linearly on TMAO concentration, and that the sensitivity of ⌬G°to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of ⌬G°on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.Keywords: Protein stability; protein folding; Notch ankyrin domain; Barnase; osmolytes Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is found in several marine organisms containing elevated intracellular urea concentrations (Robertson 1966(Robertson , 1975Griffith et al. 1974). Numerous studies have investigated the effect of TMAO on proteins and described its stabilizing effects (Yancey and Somero 1979;Lin and Timasheff 1994;Jaravine et al. 2000). Yancey et al. (1982) showed, by gel filtration chromatography, that TMAO promotes folding of proteins into more compact forms. They further showed, by recovery of enzymatic activity, that TMAO promotes folding to specific, biologically relevant native states (Yancey et al. 1982). Wang and Bolen (1997) provided an explanation for the ability of TMAO to promote specific refolding to the native structure, in which unfavorable thermodynamic interactions between TMAO and the peptide backbone destabilize the denatured state, shifting equilibrium toward the native state. Preferential interaction data from Lin and Timasheff (1994) are consistent with this interpretation, showing the region of solvent near the denatured state of the protein to be rarified in TMAO.The functional dependence of protein stability on TMAO has been analyzed in several systems and has led to different Reprint requ...

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of a-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N e I e U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of 1, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagenesis experiments indicate that the interactions between the A, G, and H helices in 1 are loose (Hughson, EM., Barrick, D., & Baldwin, R.L., 1991, Biochemistry30, 4113-41 18), which can explain why I is formed rapidly from U at the start of folding. These experiments are consistent with the explanation proposed earlier (Baldwin, R.L., 1989, Trends Biochem. Sci. 14, 291-294) for the stabilization of native secondary structures in molten globule intermediates, namely that each unit of native secondary structure has a hydrophobic face, and the hydrophobic surfaces of two units of secondary structure can interact loosely to provide mutual stabilization. These topics are discussed here in the light of some additional results.Keywords: apomyoglobin; energetics of folding; folding pathways; molten globule; protein folding It is now generally agreed that structured intermediates are observable during the kinetics of folding of most small proteins. If they are on-pathway intermediates, then it is possible to determine the folding pathway by characterizing their structures. The method of pulsed H/D exchange, which measures the exchange rates of individual peptide NH protons in a folding intermediate using the Abbreviations: Mb, myoglobin; apoMb, apomyoglobin; holoMb, holomyoglobin; 2D, two-dimensional; H-bond, hydrogen bond; H/D, hydrogenldeuterium; ANS, 1-anilinonaphthalene-8-sulfonic acid; or-LA, a-lactalbumin; cyt c, cytochrome c; pH,, pH midpoint of an unfolding transition; pH', pH measured in *HZO without correction for the isotope effect; GdmC1, guanidinium chloride.