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Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.

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Two novel cross-linkers, disuccinimidyl tartarate (DST) and N,N'-bis(3-succinimidyloxycarbonylpropyl)tartaramide (SPT), have been synthesized. These reagents span 6 and 18 A, respectively, between functional groups and contain a vic-glycol bond which can be cleaved with periodate under mild reaction conditions. Both DST and SPT have been used to examine the near-neighbor relationships of polypeptides in ubiquinone cytochrome c reductase (complex III) from beef heart mitochondria. Among the cross-linked products resolved were pairs containing I + II, II + VI, I + V, and VI + VII. Polypeptides III and IV, a cytochrome b aproprotein, and the cytochrome c1 hemoprotein, respectively, were also resolved in several cross-linked products.

The deuterium magnetic resonance spectra of lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1416,16,16-trideuteropalmitoyl-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The lessordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2-to 3fold decrease in relative amount from 2 to 220. The rate of exchange of lipid between the free and restricted environments is slower than 103/sec. The significance of these phenomena is discussed. There is now convincing evidence that some membrane proteins extend into and, in some cases, through the lipid bilayer (1, 2). For such proteins, the interactions of the lipid with the protein and with other lipids have an important role in establishing the architecture of the membrane and the function of the proteins included in that membrane.Recently, it has been possible to apply various forms of spectroscopy to the problem of lipid-protein interactions. In a pioneering work, Jost et al. (3) used electron spin resonance techniques to study the interaction of beef heart mitochondrial cytochrome c oxidase with the natural mixtures of mitochondrial lipids. This work demonstrated a highly restricted or immobilized component of the lipid corresponding to about 0.2 mg of lipid per mg of protein, independent of the ratio of lipid to protein. Presumably, this immobilized component is due to the interaction between the protein and the lipid. The authors named this restricted lipid "boundary lipid" and suggested that 0.2 mg/mg corresponds to a single solvation layer of lipid about the circumference of protein where it is in contact with lipid bilayer. A number of further questions concerning the nature of the boundary lipid are raised by this important study. Some examples are: Does the protein preferentially sequester some lipids as opposed to others? What is the energy difference between boundary lipid and free lipid under a given set of conditions? What is the time scale of the exchange of the boundary and free lipid?Deuterium nuclear magnetic resonance (DMR) offers a powerful technique for probing these questions (4-6). When rapid and complete reorientation of the deuterated molecule with respect to the applied magnetic fields takes place in anThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5435isotropic medium such as a typical flui...