The principles governing protein evolution under strong selection are important because of the recent history of evolved resistance to insecticides, antibiotics, and vaccines. One experimental approach focuses on studies of mutant proteins and all combinations of mutant sites that could possibly be intermediates in the evolutionary pathway to resistance. In organisms carrying each of the engineered proteins, a measure of protein function or a proxy for fitness is estimated. The correspondence between protein sequence and fitness is widely known as a fitness landscape or adaptive landscape. Here, we examine some empirical fitness landscapes and compare them with simulated landscapes in which the fitnesses are randomly assigned. We find that mutant sites in real proteins show significantly more additivity than those obtained from random simulations. The high degree of additivity is reflected in a summary statistic for adaptive landscapes known as the "roughness," which for the actual proteins so far examined lies in the smallest 0.5% tail of random landscapes.antibiotic resistance | fitness landscape | molecular evolution A ttempts to control agents of infectious disease or their vectors have been frustrated time and again by the evolution of resistance in the targeted proteins. How proteins evolve under strong selection is therefore an important line of inquiry, particularly in regard to whether evolutionary pathways can be reproduced or predicted.The modern concept of protein evolution as a kind of walk in sequence space seems to have originated with John Maynard Smith (1). Responding to a criticism of the theory of natural selection that the number of possible polypeptide sequences is so large that no functional protein could conceivably have arisen by random mutation, Maynard Smith emphasized that favorable mutations are incorporated into a protein sequentially, not simultaneously. He argued by analogy with a word game called change-one-letter, in which the object at each turn is to change one letter in a word to yield a meaningful different word. His example was sequential changes from WORD to GENE as follows: WORD → WORE → GORE → GONE → GENE. His rationale was that, in Darwinian evolution, each change in a protein sequence should be better (or at least no worse) than the present sequence. The basis of these assumptions, he argued, was "that no sensible alternatives have been suggested and that no evidence exists at the moment to invalidate them." And so it is today, despite intelligent design and other creationist critiques.One limitation of the analogy to the change-one-letter game is that it is usually unknown whether altering a particular amino acid in a protein results in a change in fitness that is beneficial, neutral, or deleterious, hence it is unclear which amino acid replacements are allowed. By means of studying a protein whose sequence can be changed experimentally, and choosing a proxy measure of fitness (such as catalytic activity, protein stability, or drug resistance), the change-one-letter an...