Biochemistry 1961

Formation of Native Ribonuclease from the Reduced Chain

C. B. Anfinsen, E. Haber, M. Sela & F. H. White Jr.

A protein's three-dimensional shape is written entirely in its own amino-acid sequence.

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In depth · the introduction

Unfold a protein until it's a useless tangle — then watch it tie itself back into exactly the right shape, all on its own.

The big idea

Proteins are long chains of amino-acid "beads" that fold into precise 3-D shapes, and the shape is what lets each protein do its job. A natural question is: who folds them? Is there a machine in the cell that bends each chain into place?

Anfinsen and his colleagues showed the answer is hidden in the chain itself. They took ribonuclease — an enzyme that cuts up RNA — completely apart, unfolding it into a limp string with no activity. Then they simply let it be. It refolded, by itself, back into its original working shape. Nothing pushed it there except its own chemistry. The instructions for the shape, it turned out, are written entirely in the order of the beads.

How it came about

In the late 1950s biologists knew proteins had specific folds, but not what set them. Ribonuclease was a good test subject: small, tough, and stitched together by four internal "staples" (disulfide bonds) joining eight sulphur-bearing amino acids. Anfinsen's group, at the U.S. National Institutes of Health, snipped all four staples and unfolded the chain in a strong chemical bath. The enzyme went dead.

Then they washed the chemicals away and let air back in. Slowly, the eight sulphur atoms found their correct partners again — out of 105 ways they could have paired up, they re-formed the one native set — and the enzyme came fully back to life. A neat twist sealed it: when they let the chain re-stitch while still unfolded, it locked into a wrong, "scrambled" tangle with almost no activity, but a pinch of the right chemical let the staples reshuffle and the protein settled back to its true shape. Anfinsen received the 1972 Nobel Prize in Chemistry for the idea.

Why it mattered

This turned protein folding into a solvable physics problem: if the sequence alone decides the shape, then in principle you could read a protein's sequence and compute its structure. That dream drove decades of work and was finally realised by AlphaFold, which predicts a protein's 3-D shape straight from its sequence. The same principle underlies designing brand-new proteins and understanding diseases where proteins fold wrong.

A way to picture it

Think of a specially weighted necklace that, no matter how you crumple it and drop it, always falls into the same knot — because that knot is its most relaxed, lowest-energy shape. A protein chain is like that: its sequence is chosen so that one particular fold is the most comfortable resting state, and the chain rolls "downhill" into it. Tangle it the wrong way and lock it, and it's stuck; loosen it just enough, and it slides back to the same final knot every time.

Where it sits

Sequence had just become readable — Sanger had spelled out insulin's amino acids in the early 1950s — and Watson and Crick's DNA showed how that sequence is stored and copied. Anfinsen supplied the next link: the sequence not only is the gene's message, it also folds itself into a working machine. That chain of ideas runs straight to today's structure-prediction tools and protein design, and to the study of misfolding diseases like Alzheimer's.

The original document

Original source text

C. B. Anfinsen, E. Haber, M. Sela & F. H. White Jr. · Proc. Natl. Acad. Sci. USA 47 (1961): 1309–1314

The question

A folded protein has a precise three-dimensional shape on which its function depends. Does the cell need special machinery to fold it, or is the shape already dictated by the chain itself? The authors test this with an enzyme they can unfold and then watch refold in a test tube.

The protein

Bovine pancreatic ribonuclease A is a small, robust enzyme of 124 amino acids. Its single chain is cross-linked by four disulfide bonds, formed between eight cysteine residues — internal staples that hold the fold together.

Unfolding it

Treating ribonuclease with a reducing agent (β-mercaptoethanol) in concentrated urea breaks all four disulfide bonds and dissolves the compact structure into a floppy, randomly coiled chain. The enzyme loses essentially all of its activity.

Letting it refold

When the urea and reducing agent are removed and the unfolded chain is exposed to air, it slowly reoxidises. Over a few hours — after a measurable lag — enzyme activity returns to nearly its full original level, as the eight cysteines find and reform the correct, native set of disulfide bonds with no outside help.

The scrambled control

Reoxidised while still held unfolded in 8 M urea, the chain instead locks into a near-random, "scrambled" mixture of disulfide bonds and shows only about one percent of normal activity. Add back a trace of reducing agent — enough to let the bonds break and reshuffle — and the molecule relaxes to the same native structure, recovering full activity.

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Eight cysteines can be joined into four disulfides in 105 different ways, only one of them native; random pairing would give about one part in a hundred. That the chain nonetheless reaches the native set essentially every time means the native fold is selected by stability, not by chance or by the order in which bonds happen to form.

The conclusion — later called the thermodynamic hypothesis, or Anfinsen's dogma — is that the information specifying a protein's native three-dimensional structure is contained in its amino-acid sequence, and the native fold is simply its most stable (lowest free-energy) state under physiological conditions.

National Institutes of Health, Bethesda · 1961