By filling all the RAM slots you could create what is known as a memory hole. I wouldn't worry about it, it's more common on the last-generation of Asus boards with older BIOS. And really if you are processing massive amounts of data like a server would, it would be something to worry about.
2x2GB is just better, maybe you'd like to upgrade later or overclock without so much heat generated in that area.
Very, very little performance difference that is not even noticeable without the use of a good RAM stressing benchmark. Either way, performance will be fine.
What brand are you planning on going with?